Quinazolinones as inhibitors of human phosphatidylinositol 3-kinase delta

ABSTRACT

Compounds that inhibit PI3Kδ activity, including compounds that selectively inhibit PI3Kδ activity, are disclosed. Methods of inhibiting phosphatidylinositol 3-kinase delta isoform (PI3Kδ) activity, and methods of treating diseases, such as disorders of immunity and inflammation in which PI3Kδ plays a role in leukocyte function, using the compounds also are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional patentapplication Ser. No. 60/570,784, filed May 13, 2004, the entiredisclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to phosphatidylinositol 3-kinase(PI3K) enzymes, and more particularly to selective inhibitors of PI3Kactivity and methods of using such inhibitors.

BACKGROUND OF THE INVENTION

Cell signaling via 3′-phosphorylated phosphoinositides has beenimplicated in a variety of cellular processes, e.g., malignanttransformation, growth factor signaling, inflammation, and immunity (seeRameh et al., J. Biol. Chem., 274:8347-8350 (1999) for a review). Theenzyme responsible for generating these phosphorylated signalingproducts is phosphatidylinositol 3-kinase (PI 3-kinase; PI3K). PI3Koriginally was identified as an activity associated with viraloncoproteins and growth factor receptor tyrosine kinases thatphosphorylates phosphatidylinositol (PI) and its phosphorylatedderivatives at the 3′-hydroxyl of the inositol ring (Panayotou et al.,Trends Cell Biol 2:358-60 (1992)).

Levels of phosphatidylinositol-3,4,5-triphosphate (PIP3), the primaryproduct of PI 3-kinase activation, increase upon treatment of cells witha variety of agonists. PI 3-kinase activation, therefore, is believed tobe involved in a range of cellular responses including cell growth,differentiation, and apoptosis (Parker et al., Curr. Biol., 5:577-99(1995); Yao et al., Science, 267:2003-05 (1995)). Though the downstreamtargets of phosphorylated lipids generated following PI 3-kinaseactivation have not been well characterized, emerging evidence suggeststhat pleckstrin-homology domain- and FYVE-finger domain-containingproteins are activated when binding to various phosphatidylinositollipids (Sternmark et al., J. Cell. Sci., 112:4175-83 (1999); Lemmon etal., Trends Cell Biol., 7:237-42 (1997)). In vitro, some isoforms ofprotein kinase C (PKC) are directly activated by PIP3, and thePKC-related protein kinase, PKB, has been shown to be activated by PI3-kinase (Burgering et al., Nature, 376:599-602 (1995)).

Presently, the PI 3-kinase enzyme family is divided into three classesbased on their substrate specificities. Class I PI3Ks can phosphorylatephosphatidylinositol (PI), phosphatidylinositol-4-phosphate, andphosphatidylinositol-4,5-biphosphate (PIP2) to producephosphatidylinositol-3-phosphate (PIP),phosphatidylinositol-3,4-biphosphate, andphosphatidylinositol-3,4,5-triphosphate, respectively. Class II PI3Ksphosphorylate PI and phosphatidylinositol-4-phosphate, whereas Class IIIPI3Ks can only phosphorylate PI.

The initial purification and molecular cloning of PI 3-kinase revealedthat it was a heterodimer consisting of p85 and p110 subunits (Otsu etal., Cell, 65:91-104 (1991); Hiles et al., Cell, 70:419-29 (1992)).Since then, four distinct Class I PI3Ks have been identified, designatedPI3K α, β, δ, and γ, each consisting of a distinct 110 kDa catalyticsubunit and a regulatory subunit. More specifically, three of thecatalytic subunits, i.e., p110α, p110β, and p110γ, each interact withthe same regulatory subunit, i.e., p85, whereas p110γ interacts with adistinct p101 regulatory subunit. As described below, the patterns ofexpression of each of these PI3Ks in human cells and tissues also aredistinct. Though a wealth of information has been accumulated on thecellular functions of PI 3-kinases in general, the roles played by theindividual isoforms are largely unknown.

Cloning of bovine p110α has been described. This protein was identifiedas related to the Saccharomyces cerevisiae protein: Vps34p, a proteininvolved in vacuolar protein processing. The recombinant p110α productwas also shown to associate with p85α, to yield a PI3K activity intransfected COS-1 cells. See Hiles et al., Cell, 70, 419-29 (1992).

The cloning of a second human p110 isoform, designated p110β, isdescribed in Hu et al., Mol. Cell. Biol., 13:7677-88 (1993). Thisisoform is said to associate with p85 in cells, and to be ubiquitouslyexpressed, as p110β mRNA has been found in numerous human and mousetissues, as well as in human umbilical vein endothelial cells, Jurkathuman leukemic T cells, 293 human embryonic kidney cells, mouse 3T3fibroblasts, HeLa cells, and NBT2 rat bladder carcinoma cells. Such wideexpression suggests that the p110β isoform is broadly important insignaling pathways.

Identification of the p110δ isoform of PI 3-kinase is described inChantry et al., J. Biol. Chem., 272:19236-41 (1997). It was observedthat the human p110δ isoform is expressed in a tissue-restrictedfashion. It is expressed at high levels in lymphocytes and lymphoidtissues, suggesting that the protein might play a role in PI3-kinase-mediated signaling in the immune system. Details concerning thep110δ isoform also can be found in U.S. Pat. Nos. 5,858,753; 5,822,910;and 5,985,589, each incorporated herein by reference. See also,Vanhaesebroeck et al., Proc. Natl. Acad. Sci. USA, 94:4330-5 (1997), andInternational Publication No WO 97/46688.

In each of the PI3Kα, β, and δ subtypes, the p85 subunit acts tolocalize PI 3-kinase to the plasma membrane by the interaction of itsSH2 domain with phosphorylated tyrosine residues (present in anappropriate sequence context) in target proteins (Rameh et al., Cell,83:821-30 (1995)). Two isoforms of p85 have been identified, p85α, whichis ubiquitously expressed, and p85β, which is primarily found in thebrain and lymphoid tissues (Volinia et al., Oncogene, 7:789-93 (1992)).Association of the p85 subunit to the PI 3-kinase p110α, β, or δcatalytic subunits appears to be required for the catalytic activity andstability of these enzymes. In addition, the binding of Ras proteinsalso upregulates PI 3-kinase activity.

The cloning of p110γ revealed still further complexity within the PI3Kfamily of enzymes (Stoyanov et al., Science, 269:690-93 (1995)). Thep110γ isoform is closely related to p110α and p110 (45-48% identity inthe catalytic domain), but as noted does not make use of p85 as atargeting subunit. Instead, p110γ contains an additional domain termed a“pleckstrinahomology domain” near its amino terminus. This domain allowsinteraction of p110γ with the By subunits of heterotrimeric G proteinsand this interaction appears to regulate its activity.

The p101 regulatory subunit for PI3Kgamma was originally cloned inswine, and the human ortholog identified subsequently (Krugmann et al.,J. Biol. Chem., 274:17152-8 (1.999)). Interaction between the N-terminalregion of p101 with the N-terminal region of p110γ appears to becritical for the PI3Kγ activation through Gβγ mentioned above.

A constitutively active PI3K polypeptide is described in InternationalPublication No. WO 96/25488. This publication discloses preparation of achimeric fusion protein in which a 102-residue fragment of p85 known asthe inter-SH2 (iSH2) region is fused through a linker region to theN-terminus of murine p110. The p85 iSH2 domain apparently is able toactivate PI3K activity in a manner comparable to intact p85 (Klippel etal., Mol. Cell. Biol., 14:2675-85 (1994)).

Thus, PI 3-kinases can be defined by their amino acid identity or bytheir activity. Additional members of this growing gene family includemore distantly related lipid and protein kinases including Vps34 TOR1,TOR2 of Saccharomyces cerevisiae (and their mammalian homologs such asFRAP and mTOR), the ataxia telangiectasia gene product (ATR), and thecatalytic subunit of DNA-dependent protein kinase (DNA-PK). Seegenerally, Hunter, Cell, 83:1-4 (1995).

PI 3-kinase also appears to be involved in a number of aspects ofleukocyte activation. A p85-associated PI 3-kinase activity has beenshown to physically associate with the cytoplasmic domain of CD28, whichis an important costimulatory molecule for the activation of T-cells inresponse to antigen (Pages et al., Nature, 369:327-29 (1994); Rudd,Immunity, 4:527-34 (1996)). Activation of T cells through CD28 lowersthe threshold for activation by antigen and increases the magnitude andduration of the proliferative response. These effects are linked toincreases in the transcription of a number of genes includinginterleukin-2 (IL2), an important T cell growth factor (Fraser et al.,Science, 251:313-16 (1991)). Mutation of CD28 such that it can no longerinteract with PI 3-kinase leads to a failure to initiate IL2 production,suggesting a critical role for PI 3-kinase in T cell activation.

Specific inhibitors against individual members of a family of enzymesprovide invaluable tools for deciphering functions of each enzyme. Twocompounds, LY294002 and wortmannin, have been widely used as PI 3-kinaseinhibitors. These compounds, however, are nonspecific PI3K inhibitors,as they do not distinguish among the four members of Class I PI3-kinases. For example, the IC₅₀ values of wortmannin against each ofthe various Class I PI 3-kinases are in the range of 1-10 nM. Similarly,the IC₅₀ values for LY294002 against each of these PI 3-kinases is about1 μM (Fruman et al., Ann. Rev. Biochem., 67:481-507 (1998)). Hence, theutility of these compounds in studying the roles of individual Class IPI 3-kinases is limited.

Based on studies using wortmannin, evidence exists that PI 3-kinasefunction also is required for some aspects of leukocyte signalingthrough G-protein coupled receptors (Thelen et al., Proc. Natl. Acad.Sci. USA, 91:4960-64 (1994)). Moreover, it has been shown thatwortmannin and LY294002 block neutrophil migration and superoxiderelease. However, because these compounds do not distinguish among thevarious isoforms of PI3K, it remains unclear which particular PI3Kisoform or isoforms are involved in these phenomena.

In view of the above considerations, it is clear that existing knowledgeis lacking with respect to structural and functional features of the PI3-kinase enzymes, including subcellular localization, activation states,substrate affinities, and the like. Moreover, the functions that theseenzymes perform in both normal and diseased tissues remains to beelucidated. In particular, the function of PI3Kδ in leukocytes has notbeen characterized previously, and knowledge concerning its function inhuman physiology remains limited. The coexpression in these tissues ofother PI3K isoforms has heretofore confounded efforts to segregate theactivities of each enzyme. Furthermore, separation of the activities ofthe various PI3K isozymes may not be possible without identification ofinhibitors that demonstrate selective inhibition characteristics.Indeed, applicants presently are not aware that such selective, orbetter, specific, inhibitors of PI3K isozymes have been demonstrated.

Thus, a need exists in the art for further structural characterizationof the PI3Kδ polypeptide. A need also exists for functionalcharacterization of PI3Kδ. Furthermore, understanding of PI3Kδ requiresfurther elaboration of the structural interactions of p110δ, both withits regulatory subunit and with other proteins in the cell. A need alsoremains for selective or specific inhibitors of PI3K isozymes, such thatthe functions of each isozyme can be better characterized. Inparticular, selective or specific inhibitors of PI3Kδ are desirable forexploring the role of this isozyme and for development ofpharmaceuticals to modulate activity of the isozyme.

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide compounds capable ofinhibiting the biological activity of human PI3Kδ. Another aspect of thepresent invention is to provide compounds that inhibit PI3Kδ selectivelycompared to the other PI3K isoforms. Still another aspect of theinvention is to provide a method of selectively modulating human PI3Kδactivity, and thereby promote medical treatment of diseases mediated byPI3Kδ dysfunction. Yet another aspect of the invention is to provide amethod of characterizing the function of human PI3K6.

Another aspect of the present invention is to provide a method ofdisrupting leukocyte function comprising contacting leukocytes with acompound that selectively inhibits phosphatidylinositol 3-kinase delta(PI3Kδ) activity in the leukocytes. The leukocytes can comprise cellsselected from the group consisting of neutrophils, B lymphocytes, Tlymphocytes, and basophils.

For example, in cases wherein the leukocytes comprise neutrophils, themethod comprises disrupting at least one neutrophil function selectedfrom the group consisting of stimulated superoxide release, stimulatedexocytosis, and chemotactic migration. Preferably, the method does notsubstantially disrupt bacterial phagocytosis or bacterial killing by theneutrophils. In cases wherein the leukocytes comprise B lymphocytes, themethod comprises disrupting proliferation of the B lymphocytes orantibody production by the B lymphocytes. In cases wherein theleukocytes comprise T lymphocytes, the method comprises disruptingproliferation of the T lymphocytes. In cases wherein the leukocytescomprise basophils, the method comprises disrupting histamine release bythe basophils.

In the present method, it is preferred that the PI3Kδ inhibitor isselective. It is preferred that the PI3Kδ inhibitor is at least about100-fold selective for inhibition of p110δ relative to p110α, at leastabout 40-fold selective relative to p110β, and at least about 10-foldselective relative to p110γ in a biochemical assay.

Compounds of the present invention are capable of inhibiting PI3Kδactivity and have a structural formula (I):

wherein

X and Y, independently, are N or CR;

Z is N—R⁷ or O;

R¹ are the same and are hydrogen, halo, or C₁₋₃alkyl;

R² and R³, independently, are hydrogen, halo, or C₁₋₃alkyl;

R⁴ is hydrogen, halo, OR^(a), CN, C₂₋₆alkynyl, C(═O)R^(a),C(═O)NR^(a)R^(b), C₃₋₆heterocycloalkyl,C₁₋₃alkyleneC₃₋₆heterocycloalkyl, OC₁₋₃alkyleneOR^(a),OC₁₋₃alkyleneNR^(a)R^(b), OC₁₋₃alkyleneC₃₋₆cycloalkyl,OC₃₋₆heterocycloalkyl, OC₁₋₃alkyleneC≡CH, or OC₁₋₃alkyleneC (═O)NR^(a)R^(b);

R⁵ is C₁₋₃alkyl, CH₂CF₃, phenyl, CH₂C≡CH, C₁₋₃alkyleneOR^(e),C₁₋₄alkyleneNR^(a)R^(b), or C₁₋₄alkyleneNHC(═O)OR^(a),

R⁶ is hydrogen, halo, or NR^(a)R^(b);

R⁷ is hydrogen or R⁶ and R⁷ are taken together with the atoms to whichthey are attached to form a five- or six-membered saturated ring;

R⁸ is C₁₋₃alkyl, halo, CF₃, or CH₂C₃₋₆heterocycloalkyl;

n is 0, 1, or 2;

R^(a) is hydrogen, C₁₋₄alkyl, or CH₂C₆H₅;

R^(b) is hydrogen or C₁₋₃alkyl; and

R^(c) is hydrogen, C₁₋₃alkyl, or halo, wherein when the R¹ groups aredifferent from hydrogen, R² and R⁴ are the same;

or a pharmaceutically acceptable salt, or prodrug, or solvate (e.g.,hydrate) thereof.

Another aspect of the present invention is to provide compounds ofstructural formula (II) and capable of inhibiting PI3Kδ activity:

wherein X, Y, Z, R¹ through R⁸, R^(a), R^(b), R^(c), and n are asdefined above,

or a pharmaceutically acceptable salt, or prodrug, or solvate (e.g.,hydrate) thereof.

Another aspect of the present invention is to provide a method oftreating a medical condition mediated by neutrophils comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of a compound of structural formulae (I) or (II). Exemplarymedical conditions that can be treated according to the method includethose conditions characterized by an undesirable neutrophil functionselected from the group consisting of stimulated superoxide release,stimulated exocytosis, and chemotactic migration. Preferably, accordingto the method, phagocytic activity or bacterial killing by neutrophilsis substantially uninhibited.

Still another aspect of the present invention is to provide a method ofdisrupting a function of osteoclasts comprising contacting osteoclastswith a compound of structural formulae (I) or (II).

Another aspect of the present invention is to provide a method ofameliorating a bone-resorption disorder in a mammal in need thereofcomprising administering to the mammal a therapeutically effectiveamount of a compound of structural formulae (I) or (II). A preferredbone-resorption disorder amenable to treatment according to the methodis osteoporosis.

Yet another aspect of the present invention is to provide a method ofinhibiting the growth or proliferation of cancer cells of hematopoieticorigin comprising contacting the cancer cells with a compound ofstructural formulae (I) or (II). The method can be advantageous ininhibiting the growth or proliferation of cancers selected from thegroup consisting of lymphomas, multiple myelomas, and leukemias.

Another aspect of the present invention is to provide a method ofinhibiting kinase activity of a PI3Kδ polypeptide comprising contactingthe PI3Kδ polypeptide with a compound of structural formulae (I) or(II).

Still another aspect of the present invention is to provide a method ofdisrupting leukocyte function comprising contacting leukocytes with acompound of structural formulae (I) or (II).

Another aspect of the present invention is to provide compounds ofstructural formulae (I) or (II) that inhibit PI3Kδ activity inbiochemical and cell-based assays, and exhibit a therapeutic benefit intreating medical conditions wherein PI3Kδ activity is excessive orundesirable.

Another aspect of the present invention is to provide pharmaceuticalcompositions comprising one or more compounds of structural formulae (I)or (II), and use of the compositions in a therapeutic treatment, whereininhibition of the PI3Kδ polypeptide, in vivo or ex vivo, provides atherapeutic benefit or is of research or diagnostic interest.

Another aspect of the present invention is to provide an article ofmanufacture for human pharmaceutical use comprising:

(a) a pharmaceutical composition comprising a compound of structuralformulae (I) or (II); and,

(b) a container, optionally further comprising a package insertproviding that the composition is useful in the treatment of a diseaseor disorder mediated by PI3Kδ activity.

Another aspect of the present invention is to provide:

(a) pharmaceutical composition comprising a compound of structuralformulae (I) or (II); and,

(b) a container, optionally further comprising a package insertproviding that the composition is useful in the treatment of abone-resorption disorder or a cancer of a hematopoietic origin.

These and other aspects and advantages of the present invention willbecome apparent from the following detailed description of the preferredembodiments, which are provided to enhance the understanding of theinvention without limiting the scope of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides compounds that selectively inhibit theactivity of PI3Kδ. The present invention further provides methods ofusing the compounds for inhibiting PI3Kδ activity, including methods ofselectively modulating the activity of the PI3Kδ isozyme in cells,especially leukocytes, osteoclasts, and cancer cells. The methodsinclude in vitro, in vivo, and ex vivo applications.

Of particular benefit are methods of using the compounds of theinvention for selectively modulating PI3Kδ activity in clinical settingsto ameliorate diseases or disorders mediated by PI3Kδ activity. Thus,treatment of diseases or disorders characterized by excessive orinappropriate PI3Kδ activity can be treated through administration ofselective modulators of PI3Kδ.

Moreover, the invention provides pharmaceutical compositions comprisinga selective PI3Kδ inhibitor of structural formulae (I) or (II). Alsoprovided are articles of manufacture comprising a selective PI3Kδinhibitor compound (or a pharmaceutical composition comprising thecompound) and instructions for using the compound. Other methods of theinvention include enabling the further characterization of thephysiological role of the isozyme.

The methods described herein benefit from the use of compounds thatselectively inhibit, and preferably specifically inhibit, the activityof PI3Kδ in cells, including cells in vitro, in vivo, or ex vivo. Cellstreated by methods of the present invention include those that expressendogenous PI3Kδ, wherein endogenous indicates that the cells expressPI3Kδ absent recombinant introduction into the cells of one or morepolynucleotides encoding a PI3Kδ polypeptide or a biologically activefragment thereof. The present methods also encompass use of cells thatexpress exogenous PI3Kδ, wherein one or more polynucleotides encodingPI3Kδ or a biologically active fragment thereof have been introducedinto the cell using recombinant procedures.

The cells can be in vivo, i.e., in a living subject, e.g., a mammal,including humans, wherein a PI3Kδ inhibitor can be used therapeuticallyto inhibit PI3Kδ activity in the subject. Alternatively, the cells canbe isolated as discrete cells or in a tissue, for ex vivo or in vitromethods. In vitro methods encompassed by the invention can comprise thestep of contacting a PI3Kδ enzyme or a biologically active fragmentthereof with an inhibitor compound of the invention. The PI3Kδ enzymecan include a purified and isolated enzyme, wherein the enzyme isisolated from a natural source (e.g., cells or tissues that normallyexpress a PI3Kδ polypeptide absent modification by recombinanttechnology) or isolated from cells modified by recombinant techniques toexpress exogenous enzyme.

Compounds of the invention potently inhibit p110δ. Potency typically isexpressed as the concentration of a compound required to achieve acertain result. The greater the potency, the less compound required toperform its intended function. In vitro potency typically is expressedin terms of IC₅₀ values measured using a dose-response assay. IC₅₀values can be measured by contacting a sensitive assay system with acompound of interest over a range of concentrations, includingconcentrations at which no or minimal effect is observed, through higherconcentrations at which partial effect is observed, to saturatingconcentrations at which a maximum effect is observed. Theoretically,such assays of the dose-response effect of inhibitor compounds can bedescribed as a sigmoidal curve expressing a degree of inhibition as afunction of concentration when plotted on a log scale. The curve alsotheoretically passes through a point at which the concentration issufficient to reduce activity of the p110δ enzyme to a level that is 50%that of the difference between minimal and maximal enzyme activityobserved in the assay. This concentration is defined as the InhibitoryConcentration at 50% inhibition or IC₅₀ value.

IC₅₀ values can be determined using either conventional biochemical(acellular) assay techniques or cell-based assay techniques well knownto those of ordinary skill in the art. An example of such an assay isprovided in the examples below. Preferably, IC₅₀ values are obtained byperforming the relevant assay at least twice, with the IC₅₀ valueexpressed as the average (arithmetic means, or “mean”) of the individualvalues obtained. More preferably, the assay is repeated from 3 to 10 (ormore) times, with the IC₅₀ value expressed as the mean of the valuesobtained. Still more preferably, the assay is performed a number oftimes sufficient to generate a statistically reliable mean IC₅₀ value,using statistical methods known to those of ordinary skill in the art.

Compounds of formulae (I) and (II) exhibit unexpectedly low IC₅₀ valuesrelative to PI3Kδ, corresponding to unexpectedly high in vitro potency.In various embodiments, compounds of formulae (I) and (II), when assayedas described in Example 14 below, exhibit PI3Kδ IC₅₀ values of less thanabout 250 nM, less than about 200 nM, less than about 150 nM, less thanabout 125 nM, less than about 100 nM, less than about 75 nM, less thanabout 50 nM, less than about 25 nM, less than about 10 nM, and in othersless than about 5 nM. In other embodiments, the compounds of theinvention exhibit IC₅₀ values from about 0.1 nM to about 5 nM.

The compounds of formulae (I) and (II) are selective PI3Kδ inhibitors.The term “selective PI3Kδ inhibitor” as used herein refers to a compoundthat inhibits the PI3Kδ isozyme more effectively than other isozymes ofthe PI3K family. A “selective PI3Kδ inhibitor” compound is understood tobe more selective for PI3Kδ than compounds conventionally andgenerically designated PI3K inhibitors, e.g., wortmannin or LY294002.Concomitantly, wortmannin and LY294002 are deemed “nonselective PI3Kinhibitors.” Moreover, compounds of the present invention selectivelynegatively regulate PI3Kδ expression or activity and possess acceptablepharmacological properties for use in the therapeutic methods of theinvention.

Accordingly, a selective inhibitor alternatively can be understood torefer to at least one compound that exhibits a 50% inhibitoryconcentration (IC₅₀) with respect to PI3Kδ that is at least about50-fold, at least about 100-fold, at least about 150-fold, at leastabout 200-fold, at least about 250-fold, at least about 300-fold, atleast about 350-fold, at least about 400-fold, at least about 500-fold,at least about 600-fold, at least about 700-fold, at least about800-fold, at least about 900-fold, or at least about 1000-fold lowerthan the IC₅₀ value for PI3K. In alternative embodiments, the termselective inhibitor can be understood to refer to at least one compoundthat exhibits an IC₅₀ with respect to PI3Kδ that is at least about5-fold, at least about 10-fold, at least about 20-fold, at least about30-fold, at least about 40-fold, at least about 50-fold, at least about60-fold, at least about 70-fold, at least about 80-fold, at least about90-fold, or at least about 100-fold lower than the IC₅₀ for PI3Kγ. Infurther embodiments, the term selective inhibitor can be understood torefer to at least one compound that exhibits an IC₅₀ with respect toPI3Kδ that is at least about 5-fold, at least about 10-fold, at leastabout 20-fold, at least about 30-fold, at least about 40-fold, at leastabout 50-fold, at least about 75-fold, at least about 100-fold, at leastabout 125-fold, at least about 150-fold, at least about 175-fold, atleast about 200-fold, at least about 250-fold, at least about 300-fold,or at least about 350-fold lower than the IC₅₀ for PI3 Kβ. The selectiveinhibitors are typically administered in an amount such that theyselectively inhibit PI3Kδ, as described above.

The most preferred compounds of the present invention, therefore, have alow IC₅₀ value vs. PI3Kδ (i.e., the compound is a potent inhibitor), andare selective with respect to inhibiting PI3Kδ relative to at least oneof PI3Kα, PI3K, and PI3Kγ.

“In vivo” means within a living subject, as within an animal or human.In this context, agents can be used therapeutically in vivo to retard oreliminate the proliferation of aberrantly replicating cells. The agentsalso can be used in vivo as a prophylactic to prevent aberrant cellproliferation or the manifestation of symptoms associated therewith.

“Ex vivo” means outside a living subject. Examples of ex vivo cellpopulations include cell cultures and biological samples such as fluidor tissue samples from humans or animals. Such samples can be obtainedby methods well known in the art. Exemplary biological fluid samplesinclude blood, cerebrospinal fluid, urine, saliva. Exemplary tissuesamples include tumors and biopsies. In this context, the presentcompounds can be in numerous applications, both therapeutic andexperimental.

The term “container” means any receptacle and closure therefor suitablefor storing, shipping, dispensing, and/or handling a pharmaceuticalproduct.

The term “package insert” means information accompanying the productthat provides a description of how to administer the product, along withthe safety and efficacy data required to allow the physician,pharmacist, or patient to make an informed decision regarding use of theproduct. For a pharmaceutical product approved for use in humans oranimals, the approving authority may specify the content of the packageinsert, and such a package insert may be referred to informally as the“label” for the product as the “label” for the product.

In one embodiment, the present invention provides a method of inhibitingleukocyte function. More particularly, the present invention providesmethods of inhibiting or suppressing functions of neutrophils and T andB lymphocytes. With respect to neutrophils, it unexpectedly has beenfound that inhibition of PI3Kδ activity inhibits functions ofneutrophils. For example, it has been observed that the compounds of thepresent invention elicit inhibition of classical neutrophil functions,such as stimulated superoxide production, stimulated exocytosis, andchemotactic migration. However, it further has been observed that amethod of the present invention permits suppression of certain functionsof neutrophils, while not substantially affecting other functions ofthese cells. For example, it has been observed that phagocytosis ofbacteria by neutrophils is not substantially inhibited by the selectivePI3Kδ inhibitor compounds of the present invention.

Thus, the present invention includes methods of inhibiting neutrophilfunctions, without substantially inhibiting phagocytosis of bacteria.Neutrophil functions suitable for inhibition according to the presentmethod include any function mediated by PI3Kδ activity or expression.Such functions include, without limitation, stimulated superoxiderelease, stimulated exocytosis or degranulation, chemotactic migration,adhesion to vascular endothelium (e.g., tethering/rolling ofneutrophils, triggering of neutrophil activity, and/or latching ofneutrophils to endothelium), transmural diapedesis, or emigrationthrough the endothelium to peripheral tissues. In general, thesefunctions can be collectively termed “inflammatory functions,” as theyare typically related to neutrophil response to inflammation. Theinflammatory functions of neutrophils can be distinguished from thebacterial killing functions exhibited by these cells, e.g., phagocytosisand killing of bacteria. Accordingly, the present invention furtherincludes methods of treating disease states in which one or more of theinflammatory functions of neutrophils are abnormal or undesirable.

The compounds of the present invention may be used to inhibit anendogenous immune response stimulated by at least one endogenous factorwithout substantially inhibiting an exogenous immune response stimulatedby at least one exogenous factor as disclosed in US 2005/0043239 A1,which is incorporated herein by reference. The compounds of the presentinvention may also be used to inhibit an endogenous immune responsestimulated by at least one endogenous factor without substantiallyinhibiting immune responsiveness, as disclosed in US 2005/0043239 A1.Accordingly, the compounds of the invention advantageously permittreatment of conditions associated with an undesirable endogenous immuneresponse stimulated by at least one endogenous factor withoutcompromising the ability to fight infection.

The compounds of the present invention may also be used to inhibitleukocyte accumulation as disclosed in US 2005/0054.614 A1. Thecompounds may also be used to inhibit leukocyte tethering to endothelialcells and to inhibit leukocyte transmigration into inflamed tissue, asdisclosed in US 2005/0043239 A1. Accordingly, the compounds of theinvention advantageously permit treatment of individuals having aninflammatory condition where leukocytes are found to be accumulating atthe site of insult or inflamed tissue.

It further has been established that PI3Kδ plays a role in thestimulated proliferation of lymphocytes, including B cells and T cells.Moreover, PI3Kδ appears to play a role in stimulated secretion ofantibodies by B cells. Selective PI3Kδ inhibitor compounds of thepresent invention have been employed to establish that these phenomenacan be abrogated by inhibition of PI3Kδ. Thus, the present inventionincludes methods of using compounds of structural formulae (I) or (II)for inhibiting lymphocyte proliferation, or for inhibiting antibodyproduction by B lymphocytes. Other methods enabled by the presentinvention include methods of treating disease states in which one ormore of these lymphocyte functions are abnormal or undesirable.

The methods of this invention can be practiced using compounds ofstructural formulae (I) or (II). The methods can be practiced using aracemic mixture of the compounds or a specific enantiomer. In preferredembodiments, the S-enantiomer of the compounds are utilized in thepresent methods.

The methods can be practiced using, for example, the following compoundsof the invention, but the invention is not limited to these compounds.Exemplary compounds include5-methyl-3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one;2-[1-(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;3-(2,6-difluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-difluoro-phenyl)-5-methyl-3H-quinazolin-4-one;3-(2,6-difluoro-phenyl)-2-[1-(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3M-quinazolin-4-one;3-(2,6-difluoro-phenyl)-5-methyl-2-[(1-[7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-3M-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;5-methyl-3-phenyl-2-[1-(7H-pyrrolo (2,3-d]pyrimidin-4-ylamino)-propyl)-3H-quinazolin-4-one; 2-[1-(2-fluoro-9h-purin-6-ylamino)-propyl]-5-methyl-3-phenyl-3 h-quinazolin-4-one;5-methyl-3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;2-[2-benzyloxy-1-(9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-2-benzyloxy-ethyl]-S-methyl-3-phenyl-3H-quinazolin-4-one;2-[2-benzyloxy-1-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;2-([2-benzyloxy-1-(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;3-(4-fluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(4-fluoro-phenyl)-5-methyl-3H-quinazolin-4-one;3-(4-fluoro-phenyl)-2-[1-(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3H-quinazolin-4-one;3-(4-fluoro-phenyl)-5-methyl-2-[1-(7H-pyrrolo[2,3-d]pyrimidin-4-ylaminio)-ethyl]-3H-quinazolin-4-one;5-methyl-3-phenyl-2-[1-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-3H-quinazolin-4-one;3-(3-fluoro-phenyl)-5-methyl-2-[1-(91-purin-6-ylamino)-ethyl]-3M-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-fluoro-phenyl)-5-methyl-3H-quinazolin-4-one;3-(3-fluoro-phenyl)-5-methyl-2-[1-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-3H-quinazolin-4-one;5-methyl-3-phenyl-2-[1-(9H-purin-6-yl)-pyrrolidin-2-yl]-3H-quinazolin-4-one;2-[2-hydroxy-1-(9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;5-methyl-3-phenyl-2-(phenyl-(9H-purin-6-ylamino)-methyl)-3H-quinazolin-4-one;2-[(2-amino-9H-purin-6-ylamino)-phenyl-methyl])-5-methyl-3-phenyl-3H-quinazolin-4-one;2-[(2-fluoro-9H-purin-6-ylamino)-phenyl-methyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;5-methyl-3-phenyl-2-[phenyl-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-methyl]-3H-quinazolin-4-one;5-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-fluoro-3-phenyl-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-chloro-3-phenyl-3H-quinazolin-4-one;[5-(5-methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-5-(9H-purin-6-ylamino)-pentyl]-carbamic acidbenzyl ester;[5-(2-amino-9H-purin-6-ylamino)-5-(5-methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-pentyl]-carbamic acid benzyl ester;[4-(5-methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-4-(9H-purin-6-ylamino)-butyl]-carbamic acidbenzyl ester;[4-(2-amino-9H-purin-6-ylamino)-4-(5-methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-butyl]-carbamic acid benzyl ester;3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[15-amino-1-(9H-purin-6-ylamino)-pentyl]-5-methyl-3-phenyl-3H-quinazolin-4-one);2-[5-amino-1-(2-amino-9H-purin-6-ylamino)-pentyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-Dimethyl-phenyl)-5-methyl-3H-quinazolin-4-one;3-(2,6-dimethyl-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;5-morpholin-4-ylmethyl-3-phenyl-2-[1-(9H-purin-6-ylanino)-ethyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl])-5-morpholin-4ylmethyl-3-phenyl-3H-quinazolin-4-one;2-[4-amino-1-(2-amino-9H-purin-6-ylamino)-butyl)-5-methyl-3-phenyl-3H-quinazolin-4-one;6-fluoro-3-phenyl-2-[1-(9K-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[1-(2-amino-H-purin-6-ylamino)-ethyl)-6-fluoro-3-phenyl-3H-quinazolin-4-one;2-(2-tert-butoxy-1-(9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;3-(3-methyl-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl)-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylaminio)-ethyl]-3-(3-methyl-phenyl)-5-methyl-3H-quinazolin-4-one;3-(3-chloro-phenyl)-5-methyl-2-(1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-chloro-phenyl)-5-methyl-3H-quinazolin-4-one;2-[1-(2-amino-9-purin-6-ylamino)-2-hydroxy-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl)-3-(3-fluoro-phenyl)-3H-quinazolin-4-one;2-(1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-difluoro-phenyl)-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylaminio)-propyl]-5-fluoro-3-phenyl-3H-quinazolin-4-one;5-chloro-3-(3-fluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-chloro-3-(3-fluoro-phenyl)-3H-quinazolin-4-one;3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-5-trifluoromethyl-3H-quinazolin-4-one;3-(2,6-difluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylaminio)-propyl]-3H-quinazolin-4-one;3-(2,6-difluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-3-(2,6-difluoro-phenyl)-5-methyl-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-difluoro-phenyl)-5-methyl-3H-quinazolin-4-one;3-(3,5-dichloro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;3-(2,6-dichloro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-dichloro-phenyl)-5-methyl-3H-quinazolin-4-one;5-chloro-3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-5-chloro-3-phenyl-3H-quinazolin-4-one;5-methyl-3-phenyl-2-[1-(9H-purin-6-ylamino)-butyl]-3H-quinazolin-4-one;2-[i-(2-amino-9H-purin-6-ylamino)-butyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;2-[1 (2-amino-9H-purin-6-ylamino)-ethyl]-3-(3,5-dichloro-phenyl)-5-methyl-3H-quinazolin-4-one;5-methyl-3-(3-morpholin-4-ylmethyl-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-3-(3-morpholin-4-ylmethyl-phenyl)-3H-quinazolin-4-one;2-[1-(5-bromo-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;5-methyl-2-[1-(5-methyl-7H-pyrrolo[[2,3-d]pyrimidin-4-ylamino)-ethyl]-3-phenyl-3H-quinazolin-4-one;2-[1-(5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;2-[2-hydroxy-1-(9H-purin-6-ylamino)-ethyl]-3-phenyl-3H-quinazolin-4-one;3-(3,5-difluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-3-(3,5-difluoro-phenyl)-5-methyl-3H-quinazolin-4-one;3-(3,5-difluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[1-(5-bromo-7H-pyrrolo[(2,3-d]pyrimidin-4-ylamino)-ethyl]-3-(3-fluoro-phenyl)-5-methyl-3H-quinazolin-4-one;3-(3-fluoro-phenyl)-5-methyl-2-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-3H-quinazolin-4-one;3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3,5-difluoro-phenyl)-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-propyl])-3-phenyl-3H-quinazolin-4-one;6,7-difluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;6-fluoro-3-(3-fluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[4-diethylamino-1-(9H-purin-6-ylamino)-butyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;5-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one;3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;6-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;3-(3,5-difluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;5-fluoro-2-[1-(2-fluoro-9H-purin-6-ylamino)-ethyl]-3-phenyl-3H-quinazolin-4-one;3-(3-fluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;5-chloro-3-(3,5-difluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one;3-(2,6-difluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;3-(2,6-difluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;5-Methyl-3-phenyl-2-[3,3,3-trifluoro-1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one;3-(3-hydroxy-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;3-(3-methoxy-phenyl)-5-methyl-2-{1-[9H-purin-6-ylamino]-ethyl}-3H-quinazolin-4-one;3-[3-(2-dimethylamino-ethoxy)-phenyl]-5-methyl-2-(1-[9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;3-(3-cyclopropylmethoxy-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino]-ethyl)-3H-quinazolin-4-one;5-methyl-3-(3-prop-2-ynyloxy-phenyl)-2-{1-[9H-purin-6-ylamino]-ethyl}-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino]ethyl)-3-(3-hydroxyphenyl)-5-methyl-3H-quinazolin-4-one;2-(1-[2-amino-9H-purin-6-ylamino]ethyl)-3-(3-methoxyphenyl)-5-methyl-3H-quinazolin-4-one;2-{1-[2-amino-9H-purin-6-ylamino]ethyl}-3-(3-cyclopropylmethoxy-phenyl)-5-methyl-3H-quinazolin-4-one;2-(1-(2-amino-9H-purin-6-ylamino]ethyl)-5-methyl-3-(3-prop-2-ynyloxy-phenyl)-3H-quinazolin-4-one;3-(3-ethynyl-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl)-3H-quinazolin-4-one;3-(5-methyl-4-oxo-2-[1-(9H-purin-6-ylamino)-ethyl]-4H-quinazolin-3-yl)-benzonitrile;3-(5-methyl-4-oxo-2-(1-[9H-purin-6-ylamino)-ethyl]-4H-quinazolin-3-yl)-benzamide;3-(3-acetyl-phenyl)-5-methyl-2-(1-[9H-purin-6-ylamino]-ethyl)-3H-quinazolin-4-one;2-(3-(5-methyl-4-oxo-2-[1-9H-purin-6-ylamino]-ethyl)-4H-quinazolin-3-yl-phenoxyacetamide;5-methyl-2-{1-[9H-purin-6-ylamino]-ethyl}-3-[3-(tetrahydropuran-4-yloxy)-phenyl]-3H-quinazolin-4-one;3-[3-(2-methoxy-ethoxy)-phenyl]-5-methyl-2-(1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;6-fluoro-2-[1-(9H-purin-6-ylamino)ethyl]-3-(3-(tetrahydro-pyran-4-yloxy)-phenyl)-3H-quinazolin-4-one;3-[3-(3-dimethylamino-propoxy)-phenyl]-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-ethynyl-phenyl)-5-methyl-3H-quinazolin-4-one;3-(2-[1-(2-amino-9H-purin-6-ylanino)-ethyl]-5-methyl-4-oxo-4H-quinazolin-3-yl)-benzonitrile;3-[2-[1-(2-amino-9H-purin-6-ylamino]-ethyl]-5-methyl-4-oxo-4H-quinazolin-3-yl)-benzamide;3-(2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-4-oxo-4H-quinazolin-3-yl)-benzamide;5-methyl-3-(3-morpholin-4-yl-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl)-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-3-(3-morpholin-4-yl-phenyl)-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-[3-(2-methoxy-ethoxy)-phenyl]-5-methyl-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-(2-dimethylamino-ethoxy)-phenyl]-5-methyl-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-but-3-ynyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;2-(1-(2-amino-9H-purin-6-ylamino)-but-3-ynyl]-5-methyl-3-phenyl-3H-quinazolin-4-one;5-chloro-3-(3,5-difluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-5-chloro-3-(3,5-difluoro-phenyl)-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-chloro-3-(3,5-difluoro-phenyl)-3H-quinazolin-4-one;3-(3,5-difluoro-phenyl)-6-fluoro-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one;5-chloro-3-(2,6-difluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one;2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-5-chloro-3-(2,6-difluoro-phenyl)-3H-quinazolin-4-one;5-methyl-3-phenyl-2-(1-(9H-purin-6-yloxy)-ethyl]-3H-quinazolin-4-one.

The methods of the invention can be practiced using compounds thatexhibit PI3Kδ inhibitory activity. In particular, the methods of theinvention can be practiced using compounds having the general structuralformula (I):

wherein

X and Y, independently, are N or CRC;

Z is N—R⁷ or O;

R¹ are the same and are hydrogen, halo, or C₁₋₃alkyl;

R² and R³, independently, are hydrogen, halo, or C₁₋₃alkyl;

R⁴ is hydrogen, halo, OR^(a), CN, C₂₋₆alkynyl, C(═O) R^(a), C(═O)NR^(a)R^(b), C₃₋₆heterocycloalkyl, C₁₋₃alkyleneC₃₋₆heterocycloalkyl,OC₁₋₃alkyleneOR^(a), OC₁₋₃alkyleneNR^(a)R^(b),OC₁₋₃alkyleneC₃₋₆cycloalkyl, OC₃₋₆heterocycloalkyl, OC₁₋₃alkyleneC≡CH,or OC₁₋₃alkyleneC(═O) NR^(a)R^(b);

R⁵ is C₁₋₃alkyl, CH₂CF₃, phenyl, CH₂C≡CH, C₁₋₃alkyleneOR^(c),C₁₋₃alkyleneNR^(a)R^(b), or C₁₋₄alkyleneNHC(═O)OR^(a),

R⁶ is hydrogen, halo, or NR^(a)R^(b);

R⁷ is hydrogen or R⁵ and R⁷ are taken together with the atoms to whichthey are attached to form a five- or six-membered saturated ring;

R⁸ is C₁₋₃alkyl, halo, CF₃, or CH₂C₃₋₆heterocycloalkyl;

n is 0, 1, or 2;

R^(a) is hydrogen, C₁₋₄alkyl, or CH₂C₆H₅;

R^(b) is hydrogen or C₁₋₃alkyl; and

R^(c) is hydrogen, C₁₋₃alkyl, or halo, wherein when the R¹ groups aredifferent from hydrogen, R² and R⁴ are the same;

or a pharmaceutically acceptable salt, or prodrug, or solvate (e.g.,hydrate) thereof.

Compounds of the present invention are selective inhibitors of PI3Kδactivity. The compounds exhibit inhibition of PI3Kδ in biochemicalassays, and selectively disrupt function of PI3Kδ-expressing cells incell-based assays. As described herein, the present compounds havedemonstrated an ability to inhibit certain functions in neutrophils andother leukocytes, as well as functions of osteoclasts.

In general, compounds of the present invention have the generalstructural formulae (I) or (II), or a pharmaceutically acceptable saltthereof, or prodrug, or solvate thereof:

wherein X, Y, Z, R¹ through R⁸, R^(a), R^(b), R^(c), and n are asdefined above.

In various embodiments exhibiting increased potency relative to othercompounds in accordance with the invention, R⁸ is C₁₋₃alkyl, F, Cl, orCF₃. Alternatively, in such embodiments, n is 0 (such that there is noR⁸ substituent).

In other embodiments exhibiting such increased potency, X and Y,independently, are N or CH. In further embodiment exhibiting increasedpotency, X is N and Y is CH. Alternatively, X and Y may also both be CH.In further embodiments exhibiting increased potency, R⁶ is hydrogen,halo, or NH₂.

Unexpectedly, potency against PI3Kδ is conserved when R¹ is the same. Instructural formulae (I) and (II), R² and R⁴ may differ provided that R¹is H. When R¹ is H, free rotation is unexpectedly permitted about thebond connecting the phenyl ring substituent to the quinazoline ring, andthe compounds advantageously do not exhibit atropisomerism (i.e.,multiple diasteromer formation is avoided). Alternatively, R² and R⁴ canbe the same such that the compounds advantageously do not exhibitatropisomerism.

As used herein, the term “alkyl” is defined as straight chained andbranched hydrocarbon groups containing the indicated number of carbonatoms, e.g., methyl, ethyl, and straight chain and branched propyl andbutyl groups.

The terms “C₁₋₃alkylene” and “C₁₋₄alkylene” are defined as hydrocarbongroups containing the indicated number of carbon atoms and one lesshydrogen than the corresponding alkyl group.

The term “C₂₋₆alkynyl” is defined as a hydrocarbon group containing theindicated number of carbon atoms and a carbon-carbon triple bond.

The term “C₃₋₆cycloalkyl” is defined as a cyclic hydrocarbon groupcontaining the indicated number of carbon atoms.

The term “C₂₋₆heterocycloalkyl” is defined similarly as cycloalkylexcept the ring contains one or two heteroatoms selected from the groupconsisting of O, NR^(a), and S.

The term “halo” is defined as fluoro, bromo, chloro, and iodo.

In preferred embodiments, Z is N—R⁷, and the bicyclic ring systemcontaining X and Y is

In other preferred embodiments, R¹ is hydrogen, fluoro, chloro, methyl,or

R² is hydrogen, methyl, chloro, or fluoro; R³ is hydrogen or fluoro; R⁶is NH₂, hydrogen, or fluoro; R⁷ is hydrogen or R⁵ and R⁷ are takentogether to form

R⁸ is methyl, trifluoromethyl, chloro, or fluoro; R⁴ is hydrogen,fluoro, chloro, OH, OCH₃, OCH₂C≡CH, O(CH₂)N(CH₃)₂, C(═O)CH₃, C≡CH, CN,C(═O)NH₂, OCH₂C(═O)NH₂, O(CH₂)₂OCH₃, O(CH₂)₂N(CH₃)₂,

and R⁵ is methyl, ethyl, propyl, phenyl, CH₂OH, CH₂OCH₂C₆H₅, CH₂CF₃,CH₂OC (CH₃)₃, CH₂C≡CH, (CH₂)₃N (C₂H₅)₂, (CH₂)₃NH₂, (CH₂)₄NH₂,(CH₂)₃NHC(═O) OCH₂C₆H₅, or (CH₂)₄NHC(═O)OCH₂C₆H₅; R^(c) is hydrogen,methyl, fluoro, or bromo; and n is 0 or 1.

It is generally accepted that biological systems can exhibit responsesthat are very sensitive to the absolute stereochemical nature ofcompounds to which they are exposed. See, E. J. Ariens, MedicinalResearch Reviews, 6:451-66 (1986); E. J. Ariens, Medicinal ResearchReviews, 7:367-87 (1987); K. W. Fowler, Handbook of Stereoisomers:Therapeutic Drugs, CRC Press, edited by Donald P. Smith, pp. 35-63(1989); and S. C. Stinson, Chemical and Engineering News, 75:38-70(1997).

Therefore, the compounds of the present invention include all possiblestereoisomers and geometric isomers of compounds of structural formula(I), and include not only racemic compounds, but also the opticallyactive isomers as well. In preferred embodiments, a compound of thepresent invention is the S-enantiomer of a compound (I), as depicted instructural formula (II).

When a compound of structural formula (I) is desired as a singleenantiomer, it can be obtained either by resolution of the final productor by stereospecific synthesis from either isomerically pure startingmaterial or use of a chiral auxiliary reagent. For example, see Z. Ma etal., Tetrahedron: Asymmetry, 8(6), pages 883-88 (1997). Resolution ofthe final product, an intermediate, or a starting material can beachieved by any suitable method known in the art. Specificstereoisomers, in particular, S-enantiomers of the compounds of theinvention, exhibit an excellent ability to inhibit kinase activity ofPI3Kδ.

The term “prodrug” as used herein refers to compounds that are rapidlytransformed in vivo to a compound having structural formulae (I) or(II), for example, by hydrolysis. Prodrug design is discussed generallyin Hardma et al. (Eds.), Goodman and Gilman's The Pharmacological Basisof Therapeutics, 9th ed., pp. 11-6 (1996). A thorough discussion ofprodrugs is provided in Higuchi et al., Prodrugs as Novel DeliverySystems, Vol. 14, ASCD Symposium Series, and in Roche (ed.),“Bioreversible Carriers in Drug Design,” American PharmaceuticalAssociation and Pergamon Press (1987).

Briefly, administration of a drug is followed by elimination from thebody or some biotransformation whereby biological activity of the drugis reduced or eliminated. Alternatively, a biotransformation process canlead to a metabolic by-product, which is itself more active or equallyactive as compared to the drug initially administered. Increasedunderstanding of these biotransformation processes permits the design ofso-called “prodrugs,” which, following a biotransformation, become morephysiologically active in their altered state. Prodrugs, therefore,encompass pharmacologically inactive compounds that are converted tobiologically active metabolites.

To illustrate, prodrugs can be converted into a pharmacologically activeform through hydrolysis of, for example, an ester or amide linkage,thereby introducing or exposing a functional group on the resultantproduct. Prodrugs can be designed to react with an endogenous compoundto form a water-soluble conjugate that further enhances thepharmacological properties of the compound, for example, increasedcirculatory half-life. Alternatively, prodrugs can be designed toundergo covalent modification on a functional group with, for example,glucuronic acid, sulfate, glutathione, amino acids, or acetate. Theresulting conjugate can be inactivated and excreted in the urine, orrendered more potent than the parent compound. High molecular weightconjugates also can be excreted into the bile, subjected to enzymaticcleavage, and released back into the circulation, thereby effectivelyincreasing the biological half-life of the originally administeredcompound.

Methods for Identifying Negative Regulators of PI3Kδ Activity

The PI3Kδ protein, as well as fragments thereof possessing biologicalactivity, can be used for screening putative inhibitor compounds in anyof a variety of drug screening techniques. A inhibitor of PI3Kδ is acompound that diminishes or abolishes the ability of PI3Kδ to performany of its biological functions. An example of such compounds is anagent that decreases the ability of a PI3Kδ polypeptide to phosphorylatephosphatidylinositol or to target appropriate structures within a cell.The selectivity of a compound that negatively regulates PI3Kδ activitycan be evaluated by comparing its activity on the PI3Kδ to its activityon other proteins. Selective inhibitors include, for example, antibodiesand other proteins or peptides that specifically bind to a PI3Kδpolypeptide, oligonucleotides that specifically bind to PI3Kδpolypeptides, and other nonpeptide compounds (e.g., isolated orsynthetic organic molecules) that specifically interact with PI3Kδpolypeptides. Inhibitors also include compounds as described above, butwhich interact with a specific binding partner of PI3Kδ polypeptides.

Presently preferred targets for the development of selective inhibitorsof PI3Kδ include, for example:

(1) cytoplasmic regions of PI3Kδ polypeptides that contact otherproteins and/or localize PI3Kδ within a cell;

(2) regions of PI3Kδ polypeptides that bind specific binding partners;

(3) regions of the PI3Kδ polypeptides that bind substrate;

(4) allosteric regulatory sites of the PI3Kδ polypeptides that can orcannot interact directly with the active site upon regulatory signal;

(5) regions of the PI3Kδ polypeptides that mediate multimerization.

For example, one target for development of modulators is the identifiedregulatory interaction of p85 with p110δ, which can be involved inactivation and/or subcellular localization of the p110δ moiety. Stillother selective modulators include those that recognize specificregulatory or PI3Kδ-encoding nucleotide sequences. Modulators of PI3Kδactivity can be therapeutically useful in treatment of a wide range ofdiseases and physiological conditions in which aberrant PI3Kδ activityis involved.

Accordingly, the invention provides methods of characterizing thepotency of a test compound as an inhibitor of PI3Kδ polypeptide, saidmethod comprising the steps of (a) measuring activity of a PI3Kδpolypeptide in the presence of a test compound; (b) comparing theactivity of the PI3Kδ polypeptide in the presence of the test compoundto the activity of the PI3Kδ polypeptide in the presence of anequivalent amount of a reference compound (e.g., a compound having aknown potency against PI3Kδ), wherein a lower activity of the PI3Kδpolypeptide in the presence of the test compound than in the presence ofthe reference indicates that the test compound is a more potentinhibitor than the reference compound, and a higher activity of thePI3Kδ polypeptide in the presence of the test compound than in thepresence of the reference indicates that the test compound is a lesspotent inhibitor than the reference compound.

The invention further provides methods of characterizing the potency ofa test compound as an inhibitor of PI3Kδ polypeptide, comprising thesteps of (a) determining an amount of a reference compound (e.g., aPI3Kδ inhibitor compound of the present invention) that inhibits anactivity of a PI3Kδ polypeptide by a reference percentage of inhibition,thereby defining a reference inhibitory amount for the referencecompound; (b) determining an amount of a test compound that inhibits anactivity of a PI3Kδ polypeptide by a reference percentage of inhibition,thereby defining a reference inhibitory amount for the test compound;(c) comparing the reference inhibitory amount for the test compound tothe reference inhibitory amount for the reference compound, wherein alower reference inhibitory amount for the test compound than for thereference compound indicates that the test compound is a more potentinhibitor than the reference compound, and a higher reference inhibitoryamount for the test compound than for the reference compound indicatesthat the test compound is a less potent inhibitor than the referencecompound. In one aspect, the method uses a reference inhibitory amountwhich is the amount of the compound than inhibits the activity of thePI3Kδ polypeptide by 50%, 60%, 70%, or 80%. In another aspect, themethod employs a reference inhibitory amount that is the amount of thecompound that inhibits the activity of the PI3Kδ polypeptide by 90%,95%, or 99%. These methods comprise determining the reference inhibitoryamount of the compounds in an in vitro biochemical assay, in an in vitrocell-based assay, or in an in vivo assay.

The invention further provides methods of identifying a inhibitor ofPI3Kδ activity, comprising the steps of (i) measuring activity of aPI3Kδ polypeptide in the presence and absence of a test compound, and(ii) identifying as a inhibitor a test compound that decreases PI3Kδactivity and that competes with a compound of the invention for bindingto PI3Kδ. Furthermore, the invention provides methods for identifyingcompounds that inhibit PI3Kδ activity, comprising the steps of (i)contacting a PI3Kδ polypeptide with a compound of the present inventionin the presence and absence of a test compound, and (ii) identifying atest compound as a inhibitor of PI3Kδ activity wherein the compoundcompetes with a compound of the invention for binding to PI3Kδ. Theinvention therefore provides a method for screening for candidateinhibitors of PI3Kδ activity and/or to confirm the mode of action ofcandidate such inhibitors. Such methods can be employed against otherPI3K isoforms in parallel to establish comparative activity of the testcompound across the isoforms and/or relative to a compound of theinvention.

In these methods, the PI3Kδ polypeptide can be a fragment of p110δ thatexhibits kinase activity, i.e., a fragment comprising the catalytic siteof p110δ. Alternatively, the PI3Kδ polypeptide can be a fragment fromthe p110δ-binding domain of p85 and provides a method to identifyallosteric modulators of PI3Kδ. The methods can be employed in cellsexpressing cells expressing PI3Kδ or its subunits, either endogenouslyor exogenously. Accordingly, the polypeptide employed in such methodscan be free in solution, affixed to a solid support, modified to bedisplayed on a cell surface, or located intracellularly. The modulationof activity or the formation of binding complexes between the PI3Kδpolypeptide and the agent being tested then can be measured.

Human PI3K polypeptides are amenable to biochemical or cell-based highthroughput screening (HTS) assays according to methods known andpracticed in the art, including melanophore assay systems to investigatereceptor-ligand interactions, yeast-based assay systems, and mammaliancell expression systems. For a review, see Jayawickreme et al., CurrOpin Biotechnol, 8:629-34 (1997). Automated and miniaturized HTS assaysalso are comprehended as described, for example, in Houston et al., CurrOpin Biotechnol, 8:734-40 (1997).

Such HTS assays are used to screen libraries of compounds to identifyparticular compounds that exhibit a desired property. Any library ofcompounds can be used, including chemical libraries, natural productlibraries, and combinatorial libraries comprising random or designedoligopeptides, oligonucleotides, or other organic compounds. Chemicallibraries can contain known compounds, proprietary structural analogs ofknown compounds, or compounds that are identified from natural productscreening.

Natural product libraries are collections of materials isolated fromnaturals sources, typically, microorganisms, animals, plants, or marineorganisms. Natural products are isolated from their sources byfermentation of microorganisms followed by isolation and extraction ofthe fermentation broths or by direct extraction from the microorganismsor tissues (plants or animal) themselves. Natural product librariesinclude polyketides, nonribosomal peptides, and variants (includingnonnaturally occurring variants) thereof. For a review, see Cane et al.,Science, 282:63-68 (1998).

Combinatorial libraries are composed of large numbers of relatedcompounds, such as peptides, oligonucleotides, or other organiccompounds as a mixture. Such compounds are relatively straightforward todesign and prepare by traditional automated synthesis protocols, PCR,cloning, or proprietary synthetic methods. Of particular interest arepeptide and oligonucleotide combinatorial libraries.

Still other libraries of interest include peptide, protein,peptidomimetic, multiparallel synthetic collection, recombinatorial, andpolypeptide libraries. For a review of combinatorial chemistry andlibraries created thereby, see Myers, Curr Opin Biotechnol, 8:701-07(1997).

Therapeutic Uses of Inhibitors of PI3Kδ Activity

The invention provides a method for selectively or specificallyinhibiting PI3Kδ activity therapeutically or prophylactically usingcompounds of the invention. The method comprises administering aselective or specific inhibitor of PI3Kδ activity to an individual inneed thereof in an amount sufficient to inhibit PI3Kδ activity. Themethod can be employed to treat humans or animals suffering from, orsubject to, a condition whose symptoms or pathology is mediated by PI3Kδexpression or activity.

“Treating” as used herein refers to preventing a disorder from occurringin an animal that can be predisposed to the disorder, but has not yetbeen diagnosed as having it; inhibiting the disorder, i.e., arrestingits development; relieving the disorder, i.e., causing its regression;or ameliorating the disorder, i.e., reducing the severity of symptomsassociated with the disorder. “Disorder” is intended to encompassmedical disorders, diseases, conditions, syndromes, and the like,without limitation.

The methods of the invention embrace various modes of treating an animalsubject, preferably a mammal, more preferably a primate, and still morepreferably a human. Among the mammalian animals that can be treated are,for example, humans; companion animals (pets), including dogs and cats;farm animals, including cattle, horses; sheep, pigs, and goats;laboratory animals, including rats, mice, rabbits, guinea pigs, andnonhuman primates; and zoo specimens. Nonmammalian animals include, forexample, birds, fish, reptiles, and amphibians.

A method of the present invention can be employed to treat subjects,therapeutically or prophylactically, suffering from, or subject to, aninflammatory disorder. One aspect of the present invention derives fromthe involvement of PI3Kδ in mediating aspects of the inflammatoryprocess. Without intending to be bound by any theory, it is theorizedthat, because inflammation involves processes typically mediated byleukocyte (e.g., neutrophils or lymphocyte) activation and chemotactictransmigration, and because PI3Kδ can mediate such phenomena,antagonists of PI3Kδ can be used to suppress injury associated withinflammation.

“Inflammatory disorder” as used herein can refer to any disease,disorder, or syndrome in which an excessive or unregulated inflammatoryresponse leads to excessive inflammatory symptoms, host tissue damage,or loss of tissue function. “Inflammatory disorder” also refers to apathological state mediated by influx of leukocytes and/or neutrophilchemotaxis.

“Inflammation” as used herein refers to a localized, protective responseelicited by injury or destruction of tissues, which serves to destroy,dilute, or wall off (sequester) both the injurious agent and the injuredtissue. Inflammation is associated with an influx of leukocytes and/orneutrophil chemotaxis. Inflammation can result from infection withpathogenic organisms and viruses, and from noninfectious means such astrauma or reperfusion following myocardial infarction or stroke, immuneresponse to foreign antigen, and autoimmune responses. Accordingly,inflammatory disorders amenable to the invention encompass disordersassociated with reactions of the specific defense system as well as withreactions of the nonspecific defense system.

As used herein, the term “specific defense system” refers to thecomponent of the immune system that reacts to the presence of specificantigens. Examples of inflammation resulting from a response of thespecific defense system include the classical response to foreignantigens, autoimmune diseases, and delayed type hypersensitivityresponse mediated by T-cells. Chronic inflammatory diseases, therejection of solid transplanted tissue and organs, e.g., kidney and bonemarrow transplants, and graft versus host disease (GVHD), are furtherexamples of inflammatory reactions of the specific defense system.

The term “nonspecific defense system” as used herein refers toinflammatory disorders that are mediated by leukocytes that areincapable of immunological memory (e.g., granulocytes, and macrophages).Examples of inflammation that result, at least in part, from a reactionof the nonspecific defense system include inflammation associated withconditions such as adult (acute) respiratory distress syndrome (ARDS) ormultiple organ injury syndromes; reperfusion injury; acuteglomerulonephritis; reactive arthritis; dermatoses with acuteinflammatory components; acute purulent meningitis or other centralnervous system inflammatory disorders such as stroke; thermal injury;inflammatory bowel disease; granulocyte transfusion associatedsyndromes; and cytokine-induced toxicity.

“Autoimmune disease” as used herein refers to any group of disorders inwhich tissue injury is associated with humoral or cell-mediatedresponses to the body's own constituents. “Allergic disease” as usedherein refers to any symptoms, tissue damage, or loss of tissue functionresulting from allergy. “Arthritic disease” as used herein refers to anydisease that is characterized by inflammatory lesions of the jointsattributable to a variety of etiologies. “Dermatitis” as used hereinrefers to any of a large family of diseases of the skin that arecharacterized by inflammation of the skin attributable to a variety ofetiologies. “Transplant rejection” as used herein refers to any immunereaction directed against grafted tissue, such as organs or cells (e.g.,bone marrow), characterized by a loss of function of the grafted andsurrounding tissues, pain, swelling, leukocytosis, and thrombocytopenia.

The therapeutic methods of the present invention include methods for thetreatment of disorders associated with inflammatory cell activation.“Inflammatory cell activation” refers to the induction by a stimulus(including, but not limited to, cytokines, antigens, or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatability antigens or cell adhesion molecules) ininflammatory cells (including but not limited to monocytes, macrophages,T lymphocytes, B lymphocytes, granulocytes (i.e., polymorphonuclearleukocytes such as neutrophils, basophils, and eosinophils), mast cells,dendritic cells, Langerhans cells, and endothelial cells). It will beappreciated by persons skilled in the art that activation of one or acombination of these phenotypes in these cells can contribute to theinitiation, perpetuation, or exacerbation of an inflammatory disorder.

Compounds of the present invention have been found to inhibit superoxiderelease by neutrophils. Superoxide is released by neutrophils inresponse to any of a variety of stimuli, including signals of infection,as a mechanism of cell killing. For example, superoxide release is knownto be induced by tumor necrosis factor alpha (TNFα), which is releasedby macrophages, mast cells, and lymphocytes upon contact with bacterialcell wall components such as lipopolysaccharide (LPS). TNFα is anextraordinarily potent and promiscuous activator of inflammatoryprocesses, being involved in activation of neutrophils and various othercell types, induction of leukocyte/endothelial cell adhesion, pyrexia,enhanced MHC class I production, and stimulation of angiogenesis.Alternatively, superoxide release can be stimulated byformyl-Met-Leu-Phe (fMLP) or other peptides blocked at the N-terminus byformylated methionine. Such peptides normally are not found ineukaryotes, but are fundamentally characteristic of bacteria, and signalthe presence of bacteria to the immune system. Leukocytes expressing thefMLP receptor, e.g., neutrophils and macrophages, are stimulated tomigrate up gradients of these peptides (i.e., chemotaxis) toward loci ofinfection. As demonstrated herein, compounds of the present inventioninhibit stimulated superoxide release by neutrophils in response toeither TNFα or fMLP. Other functions of neutrophils, includingstimulated exocytosis and directed chemotactic migration, also have beenshown to be inhibited by the PI3Kδ inhibitors of the invention.Accordingly, compounds of the present invention can be expected to beuseful in treating disorders, such as inflammatory disorders, that aremediated by any or all of these neutrophil functions.

The present invention enables methods of treating such diseases asarthritic diseases, such as rheumatoid arthritis, monoarticulararthritis, osteoarthritis, gouty arthritis, spondylitis; Behcet disease;sepsis, septic shock, endotoxic shock, gram negative sepsis, grampositive sepsis, and toxic shock syndrome; multiple organ injurysyndrome secondary to septicemia, trauma, or hemorrhage; ophthalmicdisorders, such as allergic conjunctivitis, vernal conjunctivitis,uveitis, and thyroid-associated ophthalmopathy; eosinophilic granuloma;pulmonary or respiratory disorders, such as asthma, chronic bronchitis,allergic rhinitis, ARDS, chronic pulmonary inflammatory disease (e.g.,chronic obstructive pulmonary disease), silicosis, pulmonarysarcoidosis, pleurisy, alveolitis, vasculitis, emphysema, pneumonia,bronchiectasis, and pulmonary oxygen toxicity; reperfusion injury of themyocardium, brain, or extremities; fibrosis, such as cystic fibrosis;keloid formation or scar tissue formation; atherosclerosis; autoimmunediseases, such as systemic lupus erythematosus (SLE), autoimmunethyroiditis, multiple sclerosis, some forms of diabetes, and Reynaud'ssyndrome; transplant rejection disorders such as GVHD and allograftrejection; chronic glomerulonephritis; inflammatory bowel diseases, suchas chronic inflammatory bowel disease (CIBD), Crohn's disease,ulcerative colitis, and necrotizing enterocolitis; inflammatorydermatoses, such as contact dermatitis, atopic dermatitis, psoriasis, orurticaria; fever and myalgias due to infection; central or peripheralnervous system inflammatory disorders, such as meningitis, encephalitis,and brain or spinal cord injury due to minor trauma; Sjögren's syndrome;diseases involving leukocyte diapedesis; alcoholic hepatitis; bacterialpneumonia; antigen-antibody complex mediated diseases; hypovolemicshock; Type I diabetes mellitus; acute and delayed hypersensitivity;disease states due to leukocyte dyscrasia and metastasis; thermalinjury; granulocyte transfusion-associated syndromes; andcytokine-induced toxicity.

The method can have utility in treating subjects suffering from, orsubject to, reperfusion injury, i.e., injury resulting from situationsin which a tissue or organ experiences a period of ischemia followed byreperfusion. The term “ischemia” refers to localized tissue anemia dueto obstruction of the inflow of arterial blood. Transient ischemiafollowed by reperfusion characteristically results in neutrophilactivation and transmigration through the endothelium of the bloodvessels in the affected area. Accumulation of activated neutrophils inturn results in generation of reactive oxygen metabolites, which damagecomponents of the involved tissue or organ. This phenomenon of“reperfusion injury” is commonly associated with conditions such asvascular stroke (including global and focal ischemia), hemorrhagicshock, myocardial ischemia or infarction, organ transplantation, andcerebral vasospasm. To illustrate, reperfusion injury occurs at thetermination of cardiac bypass procedures or during cardiac arrest whenthe heart, once prevented from receiving blood, begins to reperfuse. Itis expected that inhibition of PI3Kδ activity will result in reducedamounts of reperfusion injury in such situations.

With respect to the nervous system, global ischemia occurs when bloodflow to the entire brain ceases for a period. Global ischemia can resultfrom cardiac arrest. Focal ischemia occurs when a portion of the brainis deprived of its normal blood supply. Focal ischemia can result fromthromboembolytic occlusion of a cerebral vessel, traumatic head injury,edema, or brain tumor. Even if transient, both global and focal ischemiacan cause widespread neuronal damage. Although nerve tissue damageoccurs over hours or even days following the onset of ischemia, somepermanent nerve tissue damage can develop in the initial minutesfollowing the cessation of blood flow to the brain.

Ischemia also can occur in the heart in myocardial infarction and othercardiovascular disorders in which the coronary arteries have beenobstructed as a result of atherosclerosis, thrombus, or spasm.Accordingly, the invention is believed to be useful for treating cardiactissue damage, particularly damage resulting from cardiac ischemia orcaused by reperfusion injury in mammals.

In another aspect, selective PI3Kδ inhibitors of the present inventioncan be employed in methods of treating diseases of bone, especiallydiseases in which osteoclast function is abnormal or undesirable. Asshown below, compounds of the present invention inhibit osteoclastfunction in vitro. Accordingly, the use of such compounds and otherPI3Kδ selective inhibitors can be of value in treating osteoporosis,Paget's disease, and related bone resorption disorders.

In a further aspect, the present invention includes methods of usingPI3Kδ inhibitory compounds to inhibit the growth or proliferation ofcancer cells of hematopoietic origin, preferably cancer cells oflymphoid origin, and more preferably cancer cells related to or derivedfrom B lymphocytes or B lymphocyte progenitors. Cancers amenable totreatment using the method of the invention include, without limitation,lymphomas, e.g., malignant neoplasms of lymphoid and reticuloendothelialtissues, such as Burkitt's lymphoma, Hodgkins' lymphoma, non-Hodgkinslymphomas, lymphocytic lymphomas and the like; multiple myelomas;leukemias, such as lymphocytic leukemias, chronic myeloid (myelogenous)leukemias, and the like. In a preferred embodiment, the present PI3Kδinhibitory compounds can be used to inhibit or control the growth orproliferation of chronic myeloid (myelogenous) leukemia cells. Othercancer cells, of hematopoietic origin or otherwise, that express p110δalso can be treated by administration of a PI3Kδ inhibitor of thepresent invention (C. Sawyer et al., Cancer Research, 63(7), 1667-75(2003)).

In another aspect, the invention includes a method of suppressing afunction of basophils and/or mast cells, thereby enabling treatment ofdiseases or disorders characterized by excessive or undesirable basophiland/or mast cell activity. According to the method, a present compoundcan be used to selectively inhibit the expression or activity of PI3Kδin the basophils and/or mast cells. Preferably, the method employs aPI3Kδ inhibitor in an amount sufficient to inhibit stimulated histaminerelease by the basophils and/or mast cells. Accordingly, the use of apresent selective PI3Kδ inhibitors can be of value in treating diseasescharacterized by histamine release, i.e., allergic disorders, includingdisorders such as chronic obstructive pulmonary disease (COPD), asthma,ARDS, emphysema, and related disorders.

Pharmaceutical Compositions of Inhibitors of PI3Kδ Activity

A compound of the present invention can be administered as the neatchemical, but it is typical, and preferable, to administer the compoundin the form of a pharmaceutical composition or formulation. Accordingly,the present invention also provides pharmaceutical compositions thatcomprise a present modulator of PI3Kδ activity and a biocompatiblepharmaceutical carrier, adjuvant, or vehicle. The composition caninclude the PI3Kδ activity modulation either as the sole active agent orin combination with other agents, such as oligo- or polynucleotides,oligo- or polypeptides, drugs, or hormones mixed with an excipient orother pharmaceutically acceptable carriers. Carriers and otheringredients can be deemed pharmaceutically acceptable insofar as theyare compatible with other ingredients of the formulation and notdeleterious to the recipient thereof.

Techniques for formulation and administration of pharmaceuticalcompositions can be found in Remington's Pharmaceutical Sciences, 18thEd., Mack Publishing Co, Easton, Pa., 1990. The pharmaceuticalcompositions of the present invention can be manufactured using anyconventional method, e.g., mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping,melt-spinning, spray-drying, or lyophilizing processes. An optimalpharmaceutical formulation can be determined by one of skill in the artdepending on the route of administration and the desired dosage. Suchformulations can influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of the administered agent.Depending on the condition being treated, these pharmaceuticalcompositions can be formulated and administered systemically or locally.

The pharmaceutical compositions are formulated to contain suitablepharmaceutically acceptable carriers, and optionally can compriseexcipients and auxiliaries that facilitate processing of the activecompounds into preparations that can be used pharmaceutically. The modeof administration generally determines the nature of the carrier. Forexample, formulations for parenteral administration can comprise aqueoussolutions of the active compounds in water-soluble form. Carrierssuitable for parenteral administration can be selected from amongsaline, buffered saline, dextrose, water, and other physiologicallycompatible solutions. Preferred carriers for parenteral administrationare physiologically compatible buffers such as Hanks's solution,Ringer's solution, or physiologically buffered saline. For tissue orcellular administration, penetrants appropriate to the particularbarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. For preparations comprising proteins, theformulation can include stabilizing materials, such as polyols (e.g.,sucrose) and/or surfactants (e.g., nonionic surfactants), and the like.

Alternatively, formulations for parenteral use can comprise dispersionsor suspensions of the active compounds prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, and synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions can contain substances that increase the viscosity of thesuspension, such as sodium carboxymethylcellulose, sorbitol, dextran,and mixtures thereof. Optionally, the suspension also can containsuitable stabilizers or agents that increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Aqueous polymers that provide pH-sensitive solubilization and/orsustained release of the active agent also can be used as coatings ormatrix structures, e.g., methacrylic polymers, such as the EUDRAGIT®series available from Röhm America Inc. (Piscataway, N.J.). Emulsions,e.g., oil-in-water and water-in-oil dispersions, also can be used,optionally stabilized by an emulsifying agent or dispersant (surfaceactive materials; surfactants). Suspensions can contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethlyene sorbitoland sorbitan esters, microcrystalline cellulose, aluminum metahydroxide,bentonite, agar-agar, gum tragacanth, and mixtures thereof.

Liposomes containing the active agent also can be employed forparenteral administration. Liposomes generally are derived fromphospholipids or other lipid substances. The compositions in liposomeform also can contain other ingredients, such as stabilizers,preservatives, excipients, and the like. Preferred lipids includephospholipids and phosphatidyl cholines (lecithins), both natural andsynthetic. Methods of forming liposomes are known in the art. See, e.g.,Prescott (Ed.), Methods in Cell Biology, Vol. XIv, p. 33, AcademicPress, New York (1976).

Pharmaceutical compositions comprising the agent in dosages suitable fororal administration can be formulated using pharmaceutically acceptablecarriers well known in the art. Preparations formulated for oraladministration can be in the form of tablets, pills, capsules, cachets,dragees, lozenges, liquids, gels, syrups, slurries, elixirs,suspensions, or powders. To illustrate, pharmaceutical preparations fororal use can be obtained by combining the active compounds with a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Oral formulations can employ liquidcarriers similar in type to those described for parenteral use, e.g.,buffered aqueous solutions, suspensions, and the like.

Preferred oral formulations include tablets, dragees, and gelatincapsules. These preparations can contain one or excipients, whichinclude, without limitation:

a) diluents, such as sugars, including lactose, dextrose, sucrose,mannitol, or sorbitol;

b) binders, such as magnesium aluminum silicate, starch from corn,wheat, rice, potato, etc.;

c) cellulose materials, such as methylcellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose, polyvinylpyrrolidone,gums, such as gum arabic and gum tragacanth, and proteins, such asgelatin and collagen;

d) disintegrating or solubilizing agents such as cross-linked polyvinylpyrrolidone, starches, agar, alginic acid or a salt thereof, such assodium alginate, or effervescent compositions;

e) lubricants, such as silica, talc, stearic acid or its magnesium orcalcium salt, and polyethylene glycol;

f) flavorants and sweeteners;

g) colorants or pigments, e.g., to identify the product or tocharacterize the quantity (dosage) of active compound; and

h) other ingredients, such as preservatives, stabilizers, swellingagents, emulsifying agents, solution promoters, salts for regulatingosmotic pressure, and buffers.

Gelatin capsules include push-fit capsules made of gelatin, as well assoft, sealed capsules made of gelatin and a coating such as glycerol orsorbitol. Push-fit capsules can contain the active ingredient(s) mixedwith fillers, binders, lubricants, and/or stabilizers, etc. In softcapsules, the active compounds can be dissolved or suspended in suitablefluids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycol with or without stabilizers.

Dragee cores can be provided with suitable coatings such as concentratedsugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide,lacquer solutions, and suitable organic solvents or solvent mixtures.

The pharmaceutical composition can be provided as a salt of the activeagent. Salts are more soluble in aqueous or other protonic solvents thanthe corresponding free acid or base forms. Pharmaceutically acceptablesalts are well known in the art. Compounds that contain acidic moietiescan form pharmaceutically acceptable salts with suitable cations.Suitable pharmaceutically acceptable cations include, for example,alkali metal (e.g., sodium or potassium) and alkaline earth (e.g.,calcium or magnesium) cations.

Compounds of structural formula (I) and (II) that contain basic moietiescan form pharmaceutically acceptable acid addition salts with suitableacids. For example, Berge et al., J. Pharm. Sci., 66:1 (1977), describepharmaceutically acceptable salts in detail. The salts can be preparedin situ during the final isolation and purification of the compounds ofthe invention or separately by reacting a free base function with asuitable acid.

Pharmaceutically acceptable salts of compounds of the inventiongenerally are preferred in the methods of the invention. As used herein,the term “pharmaceutically acceptable salts” refers to salts orzwitterionic forms of the compounds of structural formulae (I) or (II).Suitable pharmaceutically acceptable cations include alkali metal (e.g.,sodium or potassium) and alkaline earth metal (e.g., calcium ormagnesium) cations. In addition, the pharmaceutically acceptable saltsof compounds of structural formulae (I) or (II) that contain a basiccenter are acid addition salts formed with pharmaceutically acceptableacids. Examples of acids which can be employed to form pharmaceuticallyacceptable salts include inorganic acids such as hydrochloric,hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic,maleic, succinic, malonic, and citric. Nonlimiting examples of salts ofcompounds of the invention include, but are not limited to,hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate,2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate,adipate, alginate, aspartate, benzoate, butyrate, camphorate,camphorsulfonate, citrate, digluconate, glycerolphosphate, hemisulfate,heptanoate, hexanoate, formate, succinate, malonate, fumarate, maleate,methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, picrate, pivalate, propionate, trichloroacetate,trifluoroacetate, glutamate, bicarbonate, paratoluenesulfonate,undecanoate, lactate, citrate, tartrate, gluconate, benzene sulphonate,and p-toluenesulphonate salts. In addition, available amino groupspresent in the compounds of the invention can be quaternized withmethyl, ethyl, propyl, and butyl chlorides, bromides, and iodides;dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl,myristyl, and steryl chlorides, bromides, and iodides; and benzyl andphnethyl bromides.

In light of the foregoing, any reference to compounds of the presentinvention appearing herein is intended to include compounds ofstructural formulae (I) and (II), as well as pharmaceutically acceptablesalts, solvates, quaternary derivatives, and prodrugs, thereof.

Compositions comprising a compound of the invention formulated in apharmaceutically acceptable carrier can be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Accordingly, there also is contemplated an article ofmanufacture, such as a container comprising a dosage form of a compoundof the invention and a label containing instructions for use of thecompound. Kits also are contemplated. For example, a kit can comprise adosage form of a pharmaceutical composition and a package insertcontaining instructions for use of the composition in treatment of amedical condition. In either case, conditions indicated on the label caninclude treatment of inflammatory disorders, cancer, and the like.

Methods of Administration of Inhibitors of PI3Kδ Activity

Pharmaceutical compositions comprising an inhibitor of PI3Kδ activitycan be administered to the subject by any conventional method, includingparenteral and enteral techniques. Parenteral administration modalitiesinclude those in which the composition is administered by a route otherthan through the gastrointestinal tract, for example, intravenous,intraarterial, intraperitoneal, intramedullary, intramuscular,intraarticular, intrathecal, and intraventricular injections. Enteraladministration modalities include, for example, oral, buccal,sublingual, and rectal administration. Transepithelial administrationmodalities include, for example, transmucosal administration andtransdermal administration. Transmucosal administration includes, forexample, enteral administration as well as nasal, inhalation, and deeplung administration; vaginal administration; and buccal and sublingualadministration. Transdermal administration includes passive or activetransdermal or transcutaneous modalities, including, for example,patches and iontophoresis devices, as well as topical application ofpastes, salves, or ointments. Parenteral administration also can beaccomplished using a high-pressure technique, e.g., POWDERJECT®.

Surgical techniques include implantation of depot (reservoir)compositions, osmotic pumps, and the like. A preferred route ofadministration for treatment of inflammation can be local or topicaldelivery for localized disorders such as arthritis, or systemic deliveryfor distributed disorders, e.g., intravenous delivery for reperfusioninjury or for systemic conditions such as septicemia. For otherdiseases, including those involving the respiratory tract, e.g., chronicobstructive pulmonary disease, asthma, and emphysema, administration canbe accomplished by inhalation or deep lung administration of sprays,aerosols, powders, and the like.

For the treatment of neoplastic diseases, especially leukemias and otherdistributed cancers, parenteral administration is typically preferred.Formulations of the compounds to optimize them for biodistributionfollowing parenteral administration would be desirable. The PI3Kδinhibitor compounds can be administered before, during, or afteradministration of chemotherapy, radiotherapy, and/or surgery.

Moreover, the therapeutic index of the PI3Kδ inhibitor compounds can beenhanced by modifying or derivatizing the compounds for targeteddelivery to cancer cells expressing a marker that identifies the cellsas such. For example, the compounds can be linked to an antibody thatrecognizes a marker that is selective or specific for cancer cells, sothat the compounds are brought into the vicinity of the cells to exerttheir effects locally, as previously described (see for example,Pietersz et al., Immunol. Rev., 129:57 (1992); Trail et al., Science,261:212 (1993); and Rowlinson-Busza et al., Curr. Opin. Oncol., 4:1142(1992)). Tumor-directed delivery of these compounds enhances thetherapeutic benefit by, inter alia, minimizing potential nonspecifictoxicities that can result from radiation treatment or chemotherapy. Inanother aspect, PI3Kδ inhibitor compounds and radioisotopes orchemotherapeutic agents can be conjugated to the same anti-tumorantibody.

For the treatment of bone resorption disorders or osteoclast-mediateddisorders, the PI3Kδ inhibitors can be delivered by any suitable method.Focal administration can be desirable, such as by intraarticularinjection. In some cases, it can be desirable to couple the compounds toa moiety that can target the compounds to bone. For example, a PI3Kδinhibitor can be coupled to compounds with high affinity forhydroxyapatite, which is a major constituent of bone. This can beaccomplished, for example, by adapting a tetracycline-coupling methoddeveloped for targeted delivery of estrogen to bone (Orme et al.,Bioorg. Med. Chem. Lett., 4(11):1375-80 (1994)).

To be effective therapeutically in modulating central nervous systemtargets, the agents used in the methods of the invention should readilypenetrate the blood brain barrier when peripherally administered.Compounds that cannot penetrate the blood brain barrier, however, canstill be effectively administered by an intravenous route.

As noted above, the characteristics of the agent itself and theformulation of the agent can influence the physical state, stability,rate of in vivo release, and rate of in vivo clearance of theadministered agent. Such pharmacokinetic and pharmacodynamic informationcan be collected through preclinical in vitro and in vivo studies, laterconfirmed in humans during the course of clinical trials. Thus, for anycompound used in the method of the invention, a therapeuticallyeffective dose can be estimated initially from biochemical and/orcell-based assays. Then, dosage can be formulated in animal models toachieve a desirable circulating concentration range that modulates PI3Kδexpression or activity. As human studies are conducted furtherinformation will emerge regarding the appropriate dosage levels andduration of treatment for various diseases and conditions.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe “therapeutic index,” which typically is expressed as the ratioLD₅₀/ED₅₀. Compounds that exhibit large therapeutic indices, i.e., thetoxic dose is substantially higher than the effective dose, arepreferred. The data obtained from such cell culture assays andadditional animal studies can be used in formulating a range of dosagefor human use. The dosage of such compounds lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity.

In accordance with the present invention, any effective administrationregimen regulating the timing and sequence of doses can be used.Compounds and pharmaceutical compositions suitable for use in thepresent invention include those wherein the active ingredient isadministered in an effective amount to achieve its intended purpose.More specifically, a “therapeutically effective amount” means an amountsufficient to modulate PI3Kδ expression or activity, and thereby treatan individual suffering an indication, or to alleviate the existingsymptoms of the indication. Determination of a therapeutically effectiveamount is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein.

Exemplary dosage levels for a human subject are of the order of fromabout 0.001 milligram of active agent per kilogram body weight (mg/kg)to about 1000 mg/kg. Typically, dosage units of the active agentcomprise from about 0.01 mg to about 1000 mg, preferably from about 0.1mg to about 100 mg, depending upon the indication, route ofadministration, and severity of the condition, for example. Depending onthe route of administration, a suitable dose can be calculated accordingto body weight, body surface area, or organ size. The final dosageregimen is determined by the attending physician in view of good medicalpractice, considering various factors that modify the action of drugs,e.g., the specific activity of the compound, the identity and severityof the disease state, the responsiveness of the patient, the age,condition, body weight, sex, and diet of the patient, and the severityof any infection. Additional factors that can be taken into accountinclude time and frequency of administration, drug combinations,reaction sensitivities, and tolerance/response to therapy. Furtherrefinement of the dosage appropriate for treatment involving any of theformulations mentioned herein is done routinely by the skilledpractitioner without undue experimentation, especially in light of thedosage information and assays disclosed, as well as the pharmacokineticdata observed in human clinical trials. Appropriate dosages can beascertained through use of established assays for determiningconcentration of the agent in a body fluid or other sample together withdose response data.

The frequency of dosing depends on the pharmacokinetic parameters of theagent and the route of administration. Dosage and administration areadjusted to provide sufficient levels of the active moiety or tomaintain the desired effect. Accordingly, the pharmaceuticalcompositions can be administered in a single dose, multiple discretedoses, continuous infusion, sustained release depots, or combinationsthereof, as required to maintain desired minimum level of the agent.Short-acting pharmaceutical compositions (i.e., short half-life) can beadministered once a day or more than once a day (e.g., two, three, orfour times a day). Long acting pharmaceutical compositions might beadministered every 3 to 4 days, every week, or once every two weeks.Pumps, such as subcutaneous, intraperitoneal, or subdural pumps, can beused for continuous infusion.

The following examples are provided to further aid in understanding theinvention, and presuppose an understanding of conventional methods wellknown to those persons having ordinary skill in the art to which theexamples pertain, e.g., the construction of vectors and plasmids, theinsertion of genes encoding polypeptides into such vectors and plasmids,or the introduction of vectors and plasmids into host cells. Suchmethods are described in detail in numerous publications including, forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press (1989), Ausubel et al. (Eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994); andAusubel et al. (Eds.), Short Protocols in Molecular Biology, 4th ed.,John Wiley & Sons, Inc. (1999). The particular materials and conditionsdescribed hereunder are intended to exemplify particular aspects of theinvention and should not be construed to limit the reasonable scopethereof.

Example 1 Preparation and Purification of Recombinant PI3Kα, β, and δ

Recombinant PI3K heterodimeric complexes consisting of a p110 catalyticsubunit and a p85 regulatory subunit were overexpressed using theBAC-TO-BAC® HT baculovirus expression system (GIBCO/BRL), and thenpurified for use in biochemical assays. The four Class I PI 3-kinaseswere cloned into baculovirus vectors as follows:

p110δ: A FLAG®-tagged version of human p110δ (SEQ ID NOS: 1 and 2) (seeChantry et al., J. Biol. Chem., 272:19236-41 (1997)) was subcloned usingstandard recombinant DNA techniques into the BamH1-Xba1 site of theinsect cell expression vector pFastbac HTb (Life Technologies,Gaithersburg, Md.), such that the clone was in frame with the His tag ofthe vector. The FLAG® system is described in U.S. Pat. Nos. 4,703,004;4,782,137; 4,851,341; and 5,011,912, and reagents are available fromEastman Kodak Co.

p110α: Similar to the method used for p110δ, described above, aFLAG®-tagged version of p110α (see Volinia et al., Genomics,24(3):427-77 (1994)) was subcloned in BamH1-HindIII sites of pFastbacHTb (Life Technologies) such that the clone was in frame with the Histag of the vector.

p110β: A p110β (see Hu et al., Mol. Cell. Biol., 13:7677-88 (1993))clone was amplified from the human MARATHON® Ready spleen cDNA library(Clontech, Palo Alto Calif.) according to the manufacturer's protocolusing the following primers:

5′ Primer (SEQ ID NO: 3) 5′-GATCGAATTCGGCGCCACCATGGACTACAAGGACGACGATGACAAGTGCTTCAGTTTCATAATGCCTCC-3′ 3′ Primer (SEQ ID NO: 4)5′-GATCGCGGCCGCTTAAGATCTGTAGTCTTTCCGAACTGTGTG-3′The 5′ primer was built to contain a FLAG® tag in frame with the p110βsequence. After amplification, the FLAG®-p110β sequence was subclonedusing standard recombinant techniques into the EcoRI-Not1 sites ofpFastbac HTa (Life Technologies), such that the clone was in frame withthe His tag of the vector.

p110γ: The p110γ cDNA (see Stoyanov et al., Science, 269:690-93 (1995))was amplified from a human Marathon Ready spleen cDNA library (Clontech)according to the manufacturer's protocol using the following primers:

5′ Primer (SEQ ID NO: 5) 5′-AGAATGCGGCCGCATGGAGCTGGAGAACTATAAACAGCCC-3′3′ Primer (SEQ ID NO: 6) 5′-CGCGGATCCTTAGGCTGAATGTTTCTCTCCTTGTTTG-3′

A FLAG® tag was subsequently attached to the 5′ end of the p110γsequence and was cloned in the BamH1-Spe1 sites of pFastbac HTb (LifeTechnologies) using standard recombinant DNA techniques, with theFLAG®-110γ sequence in-frame with the His tag of the vector.

p85α: A BamH1-EcoRI fragment of FLAG®-tagged p85 cDNA (see Skolnik etal., Cell, 65:83-89 (1991)) was subcloned into the BamH1-EcoRI sites ofthe vector pFastbac dual (Life Technologies).

Recombinant baculoviruses containing the above clones were generatedusing manufacturer's recommended protocol (Life Technologies).Baculoviruses expressing His-tagged p110α, p110β, or p110δ catalyticsubunit and p85 subunit were coinfected into Sf21 insect cells. Toenrich the heterodimeric enzyme complex, an excess amount of baculovirusexpressing p85 subunit was infected, and the His-tagged p110 catalyticsubunit complexed with p85 was purified on nickel affinity column. Sincep110γ does not associate with p85, Sf21 cells were infected withrecombinant baculoviruses expressing His-tagged p110γ only. In analternate approach, p101 can be cloned into baculovirus, to permitcoexpression with its preferred binding partner p110γ.

The 72-hour post-infected Sf21 cells (3 liters) were harvested andhomogenized in a hypotonic buffer (20 mM HEPES-KOH, pH 7.8, 5 mM KCl,complete protease inhibitor cocktail (Roche Biochemicals, Indianapolis,Ind.), using a Dounce homogenizer. The homogenates were centrifuged at1,000×g for 15 min. The supernatants were further centrifuged at10,000×g for 20 min, followed by ultracentrifugation at 100,000×g for 60min. The soluble fraction was immediately loaded onto 10 mL of HITRAP®nickel affinity column (Pharmacia, Piscataway, N.J.) equilibrated with50 mL of Buffer A (50 mM HEPES-KOH, pH 7.8, 0.5 M NaCl, 10 mMimidazole). The column was washed extensively with Buffer A, and elutedwith a linear gradient of 10-500 mM imidazole. Free p85 subunit wasremoved from the column during the washing step and only theheterodimeric enzyme complex eluted at 250 mM imidazole. Aliquots ofnickel fractions were analyzed by 10% SDS-polyacrylamide gelelectrophoresis (SDS-PAGE), stained with SYPRO® Red (Molecular Probes,Inc., Eugene, Oreg.), and quantitated with STORM® Phospholmager(Molecular Dynamics, Sunnyvale, Calif.). The active fractions werepooled and directly loaded onto a 5 mL Hi-trap heparin columnpreequilibrated with Buffer B containing 50 mM HEPES-KOH, pH 7.5, 50 mMNaCl, 2 mM dithiothreitol (DTT). The column was washed with 50 mL ofBuffer B and eluted with a linear gradient of 0.05-2 M NaCL. A singlepeak containing PI3K enzyme complex eluted at 0.8 M NaCl.SDS-polyacrylamide gel analysis showed that the purified PI3K enzymefractions contained a 1:1 stoichiometric complex of p110 and p85subunits. The protein profile of the enzyme complex during heparinchromatography corresponded to that of lipid kinase activity. The activefractions were pooled and frozen under liquid nitrogen.

Examples 2-6

Because PI3Kδ is expressed at significant levels in leukocytes, it isimportant to study the effects of the PI3Kδ-selective inhibitor onleukocyte functions. Accordingly, the effects of PI3Kδ inhibition inseveral types of leukocytes were examined. Neutrophils were examined todetermine the effects that selective inhibition of PI3Kδ might elicit(Example 2, below). It surprisingly was found that selective inhibitionof PI3Kδ activity appears to be significantly associated with inhibitionof some but not all functions characteristic of activated neutrophils.In addition, the effects of PI3Kδ inhibition on B cell and T cellfunction also were tested (Examples 3-4, below). Moreover, as PI3Kδ alsois expressed in osteoclasts, the effect of PI3Kδ inhibition on thefunction of these specialized cells was studied (Example 5, below).

Example 2 Characterization of Role of PI3Kδ in Neutrophil Function

The effects of a PI3Kδ inhibitor of the invention on neutrophilfunctions such as superoxide generation, elastase exocytosis,chemotaxis, and bacterial killing can be tested.

A. Preparation of Neutrophils from Human Blood

Aliquots (8 mL) of heparinized blood from healthy volunteers are layeredon 3 mL cushions of 7.3% FICOLL® (Sigma, St. Louis, Mo.) and 15.4%HYPAQUE® (Sigma) and centrifuged at 900 rpm for 30 min at roomtemperature in a table top centrifuge (Beckman). The neutrophil-richband just above the FICOLL®-HYPAQUE® cushion is collected and washedwith Hanks' balanced salt solution (HBSS) containing 0.1% gelatin.Residual erythrocytes are removed by hypotonic lysis with 0.2% NaCl. Theneutrophil preparation is washed twice with HBSS containing 0.1% gelatinand used immediately.

B. Measurement of Superoxide Production from Neutrophils

Superoxide generation is one of the hallmarks of neutrophil activation.A variety of activators potentiate superoxide generation by neutrophils.The effect of a present PI3Kδ inhibitor on superoxide generation bythree, different agonists: TNF1α, IgG, and fMLP, each representingseparate classes of activator, is measured. Superoxide generated by theneutrophils is measured by monitoring a change in absorbance uponreduction of cytochrome C by modification of the method described byGreen et al., (pp. 14.5.1-14.5.11 in Supp. 12, Curr. Protocols Immunol.(Eds., Colligan et al.) (1994)), as follows. Individual wells of a96-well plate are coated overnight at 4° C. with 50 μL of 2 mg/mLsolution of human fibrinogen or IgG. The wells are washed with PBS andthe following reagents were added to each well: 50 μL of HBSS orsuperoxide dismutase (1 mg/mL), 50 μL of HBSS or TNF1α (50 ng/mL), 50 μLcytochrome C (2.7 mg/mL), and 100 μL of purified human neutrophilsuspension (2×10⁶ cells/mL). The plate is centrifuged for 2 min at 200rpm and absorbance at 550 nm was monitored for 2 hr. To measure therelative amounts of superoxide generated, values obtained from thesuperoxide dismutase-containing wells are subtracted from all, andnormalized to the values obtained from the wells without any inhibitor.

Compounds of the present invention inhibit TNF-induced superoxidegeneration by neutrophils in a concentration dependent manner. Inaddition, superoxide generation induced by IgG was not significantlyinhibited by compounds of the present invention.

The effect of compounds of the present invention on superoxidegeneration induced by another potent inducer, the bacterial peptideformylated-Met-Leu-Phe (fMLP), also can be studied. Like the TNF-inducedsuperoxide generation, fMLP-induced superoxide generation also isinhibited compounds of the present invention. These results show thatthe PI3Kδ inhibitor compounds of the present invention can preventstimulus specific induction of superoxide generation by neutrophils,indicating that PI3Kδ is involved in this process.

C. Measurement of Elastase Exocytosis from Neutrophils

In addition to superoxide generation, activated neutrophils also respondby releasing several proteases that are responsible for the destructionof tissues and cartilage during inflammation. As an indication ofprotease release, the effect of present compound on elastase exocytosisis measured. Elastase exocytosis is quantitated by modification of theprocedure described by Ossanna et al. (J. Clin. Invest., 77:1939-51(1986)), as follows. Purified human neutrophils (0.2×10⁶) (treated witheither DMSO or a serial dilution of a present compound in DMSO) arestimulated with fMLP in PBS containing 0.01 mg/mL cytochalasin B, 1.0 μMsodium azide (NaN₃), 5 μg/mL L-methionine and 1 μM fMLP for 90 min at37° C. in a 96-well plate. At the end of the incubation period, theplate is centrifuged for 5 min at 1000 rpm, and 90 μL of the supernatantis transferred to 10 μL of 10 mM solution of an elastase substratepeptide, MeO-suc-Ala-Ala-Pro-Val-pNA, wherein MeO-suc=methoxy-succinyl;pNA=p-nitroanilide (Calbiochem, San Diego, Calif.). Absorbance at 410 nmis monitored for 2 hr in a 96-well plate reader. To measure the relativeamounts of elastase excytosed, all absorbance values are normalized tothe values without any inhibitor. PI3Kδ, inhibitor compounds of thepresent invention inhibit fMLP-induced elastase exocytosissignificantly, and do so in a dose-dependent fashion.

D. Measurement of fMLP-Induced Human Neutrophil Migration

Neutrophils have the intrinsic capacity to migrate through tissues, andare one of the first cell types to arrive at the sites of inflammationor tissue injury. The effect of the present compounds on neutrophilmigration towards a concentration gradient of fMLP is measured. The daybefore the migration assays are performed, 6-well plates are coated withrecombinant ICAM-1/Fc fusion protein (Van der Vieren et al., Immunity,3:683-90 (1995)) (25 μg/mL in bicarbonate buffer, pH 9.3) and leftovernight at 4° C. After washing, 1% agarose solution, in RPMI-1640 with0.5% bovine serum albumin (BSA), is added to wells with or without aninhibitor, and plates are placed into a refrigerator before punchingholes in the gelled agarose to create plaques (1 central hole surroundedby 6 peripheral ones per well).

Human neutrophils are obtained as described above, and resuspended inRPMI medium supplemented with 0.5% BSA at 5×10⁶ cells/mL. Aftercombining equal volumes of neutrophil suspension and medium (either withDMSO or a serial dilution of the test compound in DMSO), neutrophils arealiquoted into the peripheral holes, while the central hole receivedfMLP (5 μM). Plates are incubated at 37° C. in the presence of 5% CO₂for 4 hr, followed by termination of migration by the addition of 1%glutaraldehyde solution in D-PBS. After removing the agarose layer,wells are washed with distilled water and dried.

Analysis of neutrophil migration is conducted on a Nikon DIAPHOT®inverted microscope (lx objective) video workstation using the NIH 1.61program. Using Microsoft Excel and Table Curve 4 (SSPS Inc., ChicagoIll.) programs, a migration index is obtained for each of the studiedconditions. Migration index is defined as the area under a curverepresenting number of migrated neutrophils versus the net distance ofmigration per cell.

PI3Kδ inhibitor compounds of the present invention have an effect onneutrophil migration, inhibiting this activity in a dose-dependentmanner.

E. Measurement of Bactericidal Capacity of Neutrophils

Given that the PI3Kδ inhibitor compounds of the present invention affectcertain neutrophil functions, whether the compounds affectneutrophil-mediated bacterial killing is of interest. The effect of thecompounds on neutrophil-mediated Staphylococcus aureus killing isstudied according to the method described by Clark and Nauseef (pp.7.23.4-7.23.6 in Vol. 2, Supp. 6, Curr. Protocols Immunol. (Eds.,Colligan et al.) (1994)). Purified human neutrophils (5×10⁶ cells/mL)(treated with either DMSO or a serial dilution of present compound inDMSO) are mixed with autologous serum. Overnight-grown S. aureus cellsare washed, resuspended in HBSS, and added to the serum-opsonizedneutrophils at a 10:1 ratio. Neutrophils are allowed to internalize thebacteria by phagocytosis by incubation at 37° C. for 20 min. Thenoninternalized bacteria are killed by 10 units/mL lysostaphin at 37° C.for 5 min and the total mixture is rotated at 37° C. Samples arewithdrawn at various times for up to 90 min and the neutrophils arelysed by dilution in water. Viable bacteria are counted by platingappropriate dilutions on trypticase-soy-agar plate and counting the S.aureus colonies after overnight growth.

Neutrophil-mediated killing of S. aureus is similar in samples treatedwith DMSO (control) and with a present compound. Therefore, a PI3Kδinhibitor does not significantly affect the ability of neutrophils tokill S. aureus, suggesting that PI3Kδ is not involved in this pathway ofneutrophil function.

Example 3 Characterization of Role of PI3Kδ in B Lymphocyte Function

The effects of a PI3-kinase inhibitor on B cell functions includingclassical indices such as antibody production and specificstimulus-induced proliferation also are studied.

A. Preparation and Stimulation of B Cells from Peripheral Human Blood

Heparinized blood (200 mL) from healthy volunteers is mixed with anequal volume of D-PBS, layered on 10×10 mL FICOLL-PAQUE® (Pharmacia),and centrifuged at 1600 rpm for 30 min at room temperature. Peripheralblood mononuclear cells (PBMC) are collected from the FICOLLe/seruminterface, overlayed on 10 mL fetal bovine serum (FBS) and centrifugedat 800 rpm for 10 min to remove platelets. After washing, cells areincubated with DYNAL® Antibody Mix (B cell kit) (Dynal Corp., LakeSuccess, NY) for 20 min at 4-8° C. Following the removal of unboundantibody, PBL are mixed with anti-mouse IgG coated magnetic beads(Dynal) for 20 min at 4-8° C. with gentle shaking followed byelimination of labeled non-B cells on the magnetic bead separator. Thisprocedure is repeated once more. The B cells are resuspended inRPMI-1640 with 10% FBS, and kept on ice until further use.

B. Measurement of Antibody Production by Human B Cells

To study antibody production, B cells are aliquoted at 50-75×10³cells/well into 96-well plate with or without inhibitor, to which IL-2(100 U/mL) and PANSORBINe (Calbiochem) Staphylococcus aureus cells(1:90,000) were added. Part of the media is removed after 24-36 hr, andfresh media (with or without inhibitor) and IL-2 is added. Cultures areincubated at 37° C., in the presence of a CO₂ incubator for additional 7days. Samples from each condition (in triplicate) are removed, andanalyzed for IgG and IgM, as measured by ELISA. Briefly, IMMULON® 496-well plates are coated (50 μL/well) with either 150 ng/mL donkeyantihuman IgG (H+L) (Jackson ImmunoResearch, West Grove Pa.), or 2 μg/mLdonkey antihuman IgG+IgM (H+L) (Jackson ImmunoResearch) in bicarbonatebuffer, and left overnight at 4° C. After washing three times withphosphate buffered saline containing 0.1% TWEEN®-80 (PBST) (350μL/well), and blocking with 3% goat serum in PBST (100 μL/well) for 1 hrat room temperature, samples (100 UL/well) of B cell spent media dilutedin PBST are added. For IgG plates the dilution range is 1:500 to1:10000, and for IgM 1:50 to 1:1000. After 1 hr, plates are exposed tobiotin-conjugated antihuman IgG (100 ng/mL) or antihuman IgM (200 ng/mL)(Jackson ImmunoResearch) for 30 min, following by streptavidin-HRP(1:20000) for 30 min, and finally, to TMB solution (1:100) with H₂O₂(1:10000) for 5 min, with 3×PBST washing between steps. Colordevelopment is stopped by H₂SO₄ solution, and plates were read on anELISA plate reader.

Compounds of the present invention inhibited antibody production.

C. Measurement of B Cell Proliferation in Response to Cell Surface IgMStimulation

In the above experiment, B cells are stimulated using PANSORBIN®. Theeffect compounds of the present invention on B cell proliferationresponse when they are stimulated through their cell surface IgM usinganti-IgM antibody also was measured. Murine splenocytes (Balb/c) areplated into 96-well microtiter plates at 2×10⁵ cells per well in 10%FBS/RPMI. Appropriate dilutions of test inhibitor in complete medium areadded to the cells and the plates are incubated for 30-60 minutes priorto the addition of stimulus. Following the preincubation with testinhibitor, an F(ab′)₂ preparation of goat antibody specific for theμ-chain of mouse IgM is added to the wells at a final concentration of25 μg/mL. The plates are incubated at 37° C. for 3 days and 1 μCi of[³H]-thymidine is added to each well for the final four hours ofculture. The plates are harvested onto fiber filters, washed, and theincorporation of radiolabel is determined using a beta counter (Matrix96, Packard Instrument Co., Downers Grove, Ill.) and expressed as countsper minute (CPM).

Compounds of the present invention inhibit anti-IgM-stimulated B cellproliferation in a dose-dependent manner. Because compounds of thepresent invention inhibit B cell proliferation, it is envisioned thatthese compounds and other PI3Kδ inhibitors could be used to suppressundesirable proliferation of B cells in clinical settings. For example,in B cell malignancy, B cells of various stages of differentiation showunregulated proliferation. Based on the results shown above, one caninfer that PI3Kδ selective inhibitors could be used to control, limit,or inhibit growth of such cells.

Example 4 Characterization of Role of PI3Kδ in T Lymphocyte Function

T cell proliferation in response to costimulation of CD3+CD28 ismeasured. T cells are purified from healthy human blood by negativeselection using antibody coated magnetic beads according to themanufacturer's protocol (Dynal) and resuspended in RPMI. The cells aretreated with either DMSO or a serial dilution of a present compound inDMSO and plated at 1×10⁵ cells/well on a 96-well plate precoated withgoat antimouse IgG. Mouse monoclonal anti-CD3 and anti-CD28 antibodiesthen are added to each well at 0.2 ng/mL and 0.2 μg/mL, respectively.The plate is incubated at 37° C. for 24 hr and [³H]-thymidine (1μCi/well) is added. After another 18-hr incubation, the cells areharvested with an automatic cell harvester, washed, and the incorporatedradioactivity was quantified.

Although the present PI3Kδ inhibitor compounds inhibited anti-CD3- andanti-CD28-induced proliferation of T cells, an effect is not as strongas an effect on B cells or on some of the functions of neutrophils.Accordingly, the present compounds are, not toxic to cells in general.

Example 5 Characterization of Role of PI3Kδ in Osteoclast Function

To analyze the effect of the present PI3Kδ inhibitor compounds onosteoclasts, mouse bone marrow cells are isolated and differentiated toosteoclasts by treating the cells with Macrophage Colony StimulatingFactor⁻¹ (mCSF⁻¹) and Osteoprotegerin Ligand (OPGL) in serum-containingmedium (aMEM with 10% heat-inactivated FBS; Sigma) for 3 days. On dayfour, when the osteoclasts had developed, the medium is removed andcells are harvested. The osteoclasts are plated on dentine slices at 10⁵cells/well in growth medium, i.e., aMEM containing 1% serum and 2% BSAwith 55 μg/mL OPGL and 10 ng/mL mCSF⁻¹. After 3 hr, the medium ischanged to 1% serum and 1% BSA, with or without osteopontin (25 μg/mL)and the PI3K inhibitors (100 nM). The medium is changed every 24 hourswith fresh osteopontin and the inhibitors. At 72 hr, the medium isremoved, and the dentine surfaces are washed with water to remove celldebris and stained with acid hematoxylin. Excess stain is washed and thepit depths are quantitated using confocal microscopy.

The present PI3-kinase inhibitors had an inhibitory effect on osteoclastfunction. Both the nonspecific inhibitors LY294002 and wortmannininhibited osteoclast activity. However, the present PI3Kδ inhibitorcompounds had a greater effect, and in some cases almost completelyinhibited osteoclast activity.

Example 6 Characterization of Role of PI3Kδ in Basophil Function

Assessment of the effect of a compound of the invention on basophilfunction is tested using a conventional histamine release assay,generally in accordance with the method described in Miura et al., J.Immunol., 162:4198-206 (1999). Briefly, enriched basophils arepreincubated with test compounds at several concentrations from 0.1 nMto 1,000 nM for 10 min at 37° C. Then, polyclonal goat antihuman IgE(0.1 μg/mL) or fMLP is added, and allowed to incubate for an additional30 min. Histamine released into the supernatant is measured using anautomated fluorometric technique.

A dose-dependent decrease in histamine release was observed for thepresent compounds when the basophils are stimulated with anti-IgE. Thissuppression of histamine release was essentially 100% at 1,000 nM. Thepresent compound did not elicit any effect when the basophils arestimulated with fMLP. For comparison, the nonselective PI3K inhibitorLY294002 is tested at 0.1 nM and 10,000 nM, showing close to 100%inhibition of histamine release at the highest concentration.

This indicates that the present inhibitors of PI3Kδ activity can be usedto suppress release of histamine, which is one of the mediators ofallergy. Since the activity of various PI 3-kinases are required forprotein trafficking, secretion, and exocytosis in many cell types, theabove suggests that histamine release by other cells, such as mastcells, also can be disrupted by PI 3-kinase delta-selective inhibitors.

Chemical Synthesis Examples

Specific nonlimiting examples of compounds of the invention are providedbelow. It is understood in the art that protecting groups can beemployed where necessary in accordance with general principles ofsynthetic chemistry. These protecting groups are removed in the finalsteps of the synthesis under basic, acidic, or hydrogenolytic conditionsreadily apparent to persons skilled in the art. By employing appropriatemanipulation and protection of any chemical functionalities, synthesisof compounds of structural formulae (I) or (II) not specifically setforth herein can be accomplished by methods analogous to the schemes anddemonstrated synthetic procedures set forth below.

Unless otherwise noted, all starting materials were obtained fromcommercial suppliers and used without further purification. Allreactions and chromatography fractions were analyzed by thin-layerchromatography (TLC) on 250 mm silica gel plates, visualized withultraviolet (UV) light or iodine (I₂) stain. Products and intermediateswere typically purified by flash chromatography or reverse-phase highperformance liquid chromatography.

The following abbreviations are used in the synthetic examples: aq(aqueous), h (hour), min (minutes), sat'd (saturated), eq (equivalents),THE (tetrahydrofuran), RT (room temperature), Et₃N (triethylamine), Zn(zinc dust metal), n-BuOH (n-butyl alcohol), n-BuLi (n-butyl lithium),t-BuOH (tertiary butyl alcohol), NaCl (sodium chloride), MgSO₄(magnesium sulfate), BOC (C(═O)OtBu), CDCl₃ (deuterated chloroform),MtBe (methyl tert-butyl ether), H₂O (water), CHCl₃ (chloroform), HCl(hydrochloric acid), MeOH (methanol), NaOH (sodium hydroxide), NaOMe(sodium methoxide), TFA (trifluoroacetic acid), K₂CO₃ (pdtassiumcarbonate), SOCl₂ (thionyl chloride), CH₂Cl₂ (methylene chloride), EtOAC(ethyl acetate), DMF (dimethylformamide), EtOH (ethanol), DMSO (dimethylsulfoxide), NaHCO₃ (sodium bicarbonate), TLC (thin layerchromatography), HPLC (high performance liquid chromatography),electrospray ionization-mass spectrometry (ESI-MS) or MS (ES), HOBT(hydroxybenzotriazole), EDC (ethyldiethylaminopropylcarbodiimide), DIEA(diisopropylethylamine), HOAc (acetic acid), ACCUFLUOR® NFSi(N-fluorobis(phenylsulfonyl)amine) and other, similar standardabbreviations are used herein.

Example 7 Preparation of Intermediate Compounds4-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine (1)

A solution of 4-chloro-5-bromo-7H-pyrrolo[2,3-d]pyrimidine (800 mg, 3.45mmol) in THF (50 mL) at −78° C. was treated with n-BuLi (1.6 M inhexane, 2.2 eq, 7.6 mmol, 4.7 mL) dropwise, and stirred for 30 minutesat the same temperature. The mixture was treated with a solution ofACCUFLUOR® NFSi (2.0 eq, 7 mmol, 2.2 g) in THF (10 mL). The reactionmixture was allowed to warm to room temperature, was stirred for 10 h,and then concentrated to dryness. The residue was dissolved in EtOAc(100 mL), washed with water (3×15 mL) and brine (15 mL), dried withNa₂SO₄, and purified by reverse phase HPLC (10×250 mm C18 Luna column,4.7 mL/min, 10-90% acetonitrile in water over 20 min) to provideintermediate compound 1. ESI-MS m/z=172.1 (MH⁺).

The reaction described above and intermediate compound 1 are shownbelow.

2-amino-6-morpholin-4-ylmethyl-N-phenyl-benzamide (2)

Intermediate compound 2 was prepared according to the procedures setforth in steps A-F below.

6-nitro-benzoic acid methyl ester (3)

Step A: A solution of 6-nitro-benzoic acid in benzene was treated withthionyl chloride (2.5 eq) and stirred at reflux for 8 h. Afterevaporation, the residue was dissolved in chloroform and then treatedwith methanol. After stirring at reflux for 3 h, the mixture wasevaporated to afford compound 3.

2-bromomethyl-6-nitro-benzoic acid methyl ester (4)

Step B: A mixture of compound 3 (2 g), N-bromosuccinimide (1.93 g), andbenzoyl peroxide (0.124 g) in carbon tetrachloride (30 mL) was stirredat reflux overnight. After cooling, solids were filtered off and thefiltrate was concentrated to yield compound 4 as a crude, yellow oil.

2-morpholin-4-ylmethyl-6-nitro-benzoic acid methyl ester (5)

Step C: A mixture of compound 4 (3.5 g, crude material), morpholine(0.99 g), potassium carbonate (2.89 g), and potassium iodide (1.66 g) inDMF (22 mL) was stirred at room temperature overnight. The reactionmixture was poured into water (30 mL) and extracted with ethyl acetate(3×30 mL). The combined organic extracts were washed with brine (30 mL),dried over sodium sulfate, and concentrated to a residue. The crudematerial was purified by flash chromatography (silica gel, 30-50% ethylacetate/hexanes) to yield compound 5 as a yellow solid.

2-morpholin-4-ylmethyl-6-nitro-benzoic acid (6)

Step D: A solution of compound 5 (0.85 g) and NaOH (0.304 g) in amixture of water (9 mL), methanol (5 mL), and THF (45 mL) was stirred atreflux for 24 h and concentrated to dryness. The residue was dissolvedin water, and the solution was cooled to 0′C and adjusted to pH 7 with10% potassium hydrogen sulfate. The mixture was concentrated to dryness,the residue was treated with methanol, and filtered to remove solids.The filtrate was concentrated to yield compound 6 as a yellow solid.

2-morpholin-4-ylmethyl-6-nitro-N-phenyl-benzamide (7)

Step E: A solution of compound 6 (0.62 g, 2.3 mmol) in THE (50 mL) wastreated with thionyl chloride (0.94 mL), stirred at room temperature for4 h, and evaporated to dryness. The residue was dissolved in THE (50 mL)and treated with aniline (0.58 mL, 6.4 mmol) and diisopropylethylamine(1.12 mL, 6.4 mmol). The reaction mixture was stirred for 4 h,concentrated, dissolved in dichloromethane (50 mL), washed withsaturated aqueous sodium bicarbonate (25 mL) and brine (25 mL), driedwith sodium sulfate, and concentrated. The crude residue of compound 7then was purified by flash chromatography (5% methanol indichloromethane).

2-amino-6-morpholin-4-ylmethyl-N-phenyl-benzamide (2)

Step F: A solution of compound 7 (0.3 g) and 10% Pd/C (30 mg) inmethanol (35 mL) was stirred under hydrogen (1 atm) for 1.5 h. Themixture was filtered through a bed of CELITE® and the filtrate wasconcentrated to provide a yellow solid product, intermediate compound 2.ESI-MS m/z=312 (MH⁺).

Steps A-F and compounds 2-7 are shown in the reaction scheme below.

3-morpholin-4-ylmethyl-phenylamine (8)

Intermediate compound 8 (shown below) was prepared according to theprocedures set forth in steps A and B.

4-(3-nitrobenzyl)morpholine (9)

Step A: A 250 mL, one-neck, round bottomed flask equipped with amagnetic stirrer and reflux condenser was charged with1-chloromethyl-3-nitrobenzene (10.0 g, 58.3 mmol), morpholine (15.0 g,175 mmol) and toluene (75 mL), and the solution was heated at reflux for2.5 h. The reaction mixture was allowed to cool to ambient temperaturethen washed with 1N aqueous sodium hydroxide (2×50 mL). The aqueouslayer was extracted with ethyl acetate (3×50 mL), and the combinedextracts were dried over sodium sulfate and concentrated under reducedpressure to afford compound 9 as an off-white solid. ¹H NMR (CDCl₃) δ(ppm) 8.22 (s, 1H), 8.12 (d, 1H, J=8.2 Hz), 7.68 (d, 1H, J=7.6 Hz), 7.49(t, 1H, J=7.9 Hz), 3.73 (m, 4H), 3:59 (s, 2H), 2.46 (m, 4H).

3-morpholin-4-ylmethyl-phenylamine (8)

Step B: A 250-mL, three-neck, round bottomed flask equipped with amechanical stirrer and reflux condensor was charged with compound 9(12.7 g, 57.4 mmol), iron powder (40.0 g, 71.7 mmol), 2N hydrochloricacid (20 mL) and ethanol (75 mL). The resulting suspension was heated atreflux for 2 h. After this time the mixture was allowed to cool andfiltered through a pad of CELITE® 521. The filtrate was concentratedunder reduced pressure, the residue diluted with water (100 mL) andbasified with solid potassium carbonate to pH 10. The mixture wasextracted with ethyl acetate (3×75 mL) and the combined organic extractswere dried over anhydrous sodium sulfate and concentrated under reducedpressure to yield compound 8 as a dark viscous oil. ¹H NMR (CDCl₃) δ(ppm) 7.09 (t, 1H, J=8.1 Hz), 6.70 (m, 2H), 6.58 (m, 1H), 3.70 (m, 4H),3.40 (bs, 2H), 2.44 (m, 4H). Intermediate compound 8 is shown below.

6-bromo-9-(2-trimethylsilanyl-ethoxymethyl)-9H-urine (10)

A 500-mL, one-neck, round bottomed flask equipped with a magneticstirrer was charged with 6-bromopurine (10.4 g, 52.0 mmol), potassiumcarbonate (21.5 g, 156 mmol), 4 Å molecular sieves (22.6 g),dimethylformamide (200 mL) and 2-(trimethylsilyl)ethoxymethyl chloride(13.2 g, 78.0 mmol). The reaction mixture was stirred for 18 h atambient temperature, then filtered with the aid of CELITE® 521.Concentration of the filtrate under high vacuum followed by columnchromatography gave a 57% yield of intermediate compound 10 as anoff-white solid m.p. 49-52° C.; ¹H NMR (DMSO-d₆) δ (ppm) 8.95 (s, 1H),8.86 (s, 1H), 3.69 (t, 2H, J=8.0 Hz), 0.93 (t, 2H, J=8.2 Hz), 0.08 (s,9H); m/z=330 (M+H). Intermediate compound 10 is shown below.

2-amino-6-bromo-9-(2-trimethylsilylethoxy)methyl-9H-purine (11)

Intermediate compound 11 was prepared using the method described abovewith respect to intermediate compound 10, but 2-amino-6-bromopurine wasused in place of 6-bromopurine. Intermediate compound 11 is shown below.

2-di-tert-butyloxycarbonylamino-6-bromo-9-(2-trimethylsilylethoxy)methyl-9H-purine(12)

A 250-mL, one-neck, round bottomed flask equipped with a magneticstirrer was purged with nitrogen and charged with intermediate compound11 (11.7 g, 34.0 mmol), di-tert-butyl dicarbonate (22.2 g, 102 mmol),DMAP (581 mg, 4.76 mmol) and anhydrous tetrahydrofuran (150 mL). Thereaction mixture was stirred for 18 h at ambient temperature and thenevaporated to dryness. Column chromatography of the resulting residueyielded intermediate compound 12 as a white solid. m.p 93-95° C.; ¹H NMR(DMSO-d₆) δ (ppm) 8.99 (s, 1H), 5.71 (s, 2H), 3.66 (t, 2H, J=8.0 Hz),1.47 (s, 18H), 0.92 (t, 2H, J=8.2 Hz); m/z=546 (M+H). Intermediatecompound 12 is shown below.

6-chloro-9-(2-trimethylsilanyl-ethoxymethyl)-9H-purine (13)

Intermediate compound 13 was prepared using the method described abovewith respect to intermediate compound 10, but 6-chloropurine was used inplace of 6-bromopurine. Intermediate compound 13 is shown below.

General Procedures

The compounds of the present invention can be prepared by the followingmethods. Additional compounds are prepared using one of the followingmethods and selecting appropriate starting materials and reagents.General procedures and specific procedures for synthesizing the presentcompounds also are set forth in U.S. Pat. No. 6,518,277, incorporatedherein by reference.

Example 8 Compound Preparation

Compounds in accordance with general formula I (shown above) have beenprepared according to the exemplary synthetic procedures describedbelow.

5-methyl-3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one(14)

Compound (14) was prepared according to the procedures set forth insteps A-D below.

2-amino-6-methyl-N-phenyl-benzamide (15)

Step A: Thionyl chloride (14.5 mL, 198 mmol) was added to a solution of2-amino-6-methylbenzoic acid (10.0 g, 66.1 mmol) in benzene (250 mL).The resulting suspension was heated to reflux and stirred overnight.After cooling, the reaction was concentrated in vacuo, and the resultingresidue was dissolved in chloroform (300 mL). Aniline (15 mL, 165 mmol)was added, and the mixture heated to reflux. After three hours, thereaction was allowed to cool and the resulting suspension was filtered.The filtrate was subjected to flash chromatography, and the crudeproduct recrystallized from isopropanol to provide compound 15 as ayellow crystalline solid. MS (ES): m/z 227 (M+H), 134. ¹H NMR (300 MHz,DMSO-d6) δ 10.26 (s, 1H), 7.75 (d, 2H, J=7.9 Hz), 7.32 (t, 2H, J=7.8Hz), 7.07 (t, 1H, J=7.3 Hz), 7.00 (t, 1H, J=7.8 Hz), 6.58 (d, 1H, J=8.1Hz), 6.46 (d, 1H, J=7.4 Hz), 4.98 (br. s, 2H), 2.21 (s, 3H). Thereaction described above and compound 15 are shown below.

[1-(5-methyl-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)propyl]-carbamicacid benzyl ester (16)

Step B: Compound 15 (1.20 g, 5.30 mmol) was combined with2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester(2.13 g, 6.36 mmol), dimethylaminopyridine (915 mg, 7.49 mmol), anddiisopropylethylamine (1.10 mL, 6.36 mmol) in toluene (15 mL). Themixture was heated to 110° C. and stirred at that temperature for 22hours. After cooling, the reaction was purified by flash chromatographyto provide the quinazolinone (16) as a pale yellow solid. MS (ES): m/z428 (M+H). ¹H NMR (300 MHz, DMSO-d6) δ 10.26 (s, 1H), 7.75 (d, 2H, J=7.9Hz), 7.32 (t, 2H, J=7.8 Hz), 7.07 (t, 1H, J=7.3 Hz), 7.00 (t, 1H, J=7.8Hz), 6.58 (d, 1H, J=8.1 Hz), 6.46 (d, 1H, J=7.4 Hz), 4.98 (br. s, 2H),2.21 (s, 3H). The reaction described above and compound 16 are shownbelow.

2-(1-amino-propyl)-5-methyl-3-phenyl-3H-quinazolin-4-one (17)

Step C: A catalytic amount of 10% Pd/C was added to a solution ofcompound 16 (691 mg, 1.62 mmol) in ethanol (8 mL). The resulting mixturewas placed under a hydrogen atmosphere (balloon pressure) and allowed tostir at ambient temperature overnight. The reaction was filtered, andthe filtrate concentrated in vacuo. The resulting crude residue waspurified by flash chromatography to afford the free amine, compound 17,as a pale yellow oil. (ES): m/z 294 (M+H), 237. ¹H NMR (300 MHz,DMSO-d6) δ 7.68 (t, 1H, J=7.7 Hz), 7.49-7.63 (m, 3H), 7.43-7.49 (m, 1H),7.36-7.43 (m, 1H), 7.19-7.34 (m, 2H), 4.03-4.19 (m, 1H), 2.72 (s, 3H),2.04 (br. s, 2H), 1.63-1.79 (m, 1H), 1.29-1.44 (m, 1H), 0.68 (t, 3H,J=7.3 Hz). The reaction described above and compound 17 are shown below.

Step C (alternative procedure): Trifluoroacetic acid was added to asolution of the Boc-protected amine compound 16 in dichloromethane. Theresulting solution was allowed to stir at ambient temperature until LCMSor TLC indicated complete consumption of starting material. The reactionwas concentrated in vacuo, and the residue purified by flashchromatography to provide the free amine 17.

5-methyl-3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one(14)

Step D: Compound 17 (100 mg, 0.341 mmol) was combined with 6-bromopurine (75 mg, 0.375 mmol) and diisopropylethylamine (65 uL, 0.375 mmol)in n-butanol (1.0 mL). The reaction was sealed, heated to 120° C., andstirred for 18 hours. The solution was allowed to cool, thenconcentrated in vacuo. The resulting residue was purified by prep HPLCto provide compound 14 as an olive solid. (ES): m/z 412 (M+H), 206. ¹HNMR (300 MHz, DMSO-d6) δ 8.96-9.06 (br. m, 1H), 8.53 (br. s, 1H), 8.51(s, 1H), 7.68 (t, 1H, J=7.8 Hz), 7.45-7.62 (m, 6H), 7.31 (d, 1H, J=7.3Hz), 4.76-4.86 (m, 1H), 2.73 (s, 3H), 1.98-2.11 (m, 1H), 1.76-1.94 (m,1H), 0.79 (t, 3H, J=7.2 Hz). The reaction described above and compound14 are shown below.

2-[1-(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(18)

Compound 18 was prepared using the general procedure described abovewith respect to compound 14, but 2-fluoro-6-chloropurine was used inplace of 6-bromopurine in step D. ESI-MS m/z 416.1 (MH⁺). Compound 18 isshown below.

3-(2,6-difluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(19)

Compound 19 was prepared using the general procedure described abovewith respect to compound 14, but 2,6-difluorolaniline was used in placeof aniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ESI-MS m/z 434.1 (MH⁺). Compound 19 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-difluoro-phenyl)-5-methyl-3H-quinazolin-4-one(20)

Compound 20 was prepared using the general procedure described abovewith respect to compound 14, but 2,6-difluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.ESI-MS m/z 449.5 (MH⁺). Compound 20 is shown below.

3-(2,6-difluoro-phenyl)-2-[1-(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3H-quinazolin-4-one(21)

Compound 21 was prepared using the general procedure described abovewith respect to compound 14, but 2,6-difluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-fluoro-6-chloropurine was substituted for 6-bromopurine in step D.¹HNMR (MeOH-d4): δ 8.22-8.17 (m, 1H); 7.76-7.62 (m, 2H); 7.47-7.45 (m,1H); 7.38-7.33 (m, 1.2H); 7.30-7.17 (m, 1H); 6.88-6.75 (m, 1H);5.30-5.27 (m, 0.5H); 5.09-5.07 (m, 0.5H); 2.778 (s, 3H); 1.62-1.50 (m,3H). Compound 21 is shown below.

3-(2,6-difluoro-phenyl)-5-methyl-2-[1-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-3H-quinazolin-4-one(22)

Compound 22 was prepared using the general procedure described abovewith respect to compound 14, but 2,6-difluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was substituted for6-bromopurine in step D. ESI-MS m/z 433 (MH⁺). Compound 22 is shownbelow.

2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(23)

Compound 23 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-6-bromopurine was substitutedfor 6-bromopurine in step D. MS (ES): m/z 427 (M+H), 214. Compound 23 isshown below.

5-methyl-3-phenyl-2-[1-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-propyl]-3H-quinazolin-4-one(24)

Compound 24 was prepared using the general procedure described abovewith respect to compound 14, but 7-deaza-6-chloropurine was substitutedfor 6-bromopurine in step D. MS (ES): m/z 411 (M+H), 206. Compound 24 isshown below.

2-[1-(2-fluoro-9H-purin-6-ylamino)-propyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(25)

Compound 25 was prepared using the general procedure described abovewith respect to compound 14, but 2-fluoro-6-chloropurine was substitutedfor 6-bromopurine in step D. MS (ES): m/z 430 (M+H), 446. Compound 25 isshown below.

5-methyl-3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(26)

Compound 26 was prepared using the general procedure described abovewith respect to compound 14, but 2-tert-butoxycarbonylamino-propionicacid 2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. MS (ES): m/z 398 (M+H). Compound 26 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(27)

Compound 27 was prepared using the general procedure described abovewith respect to compound 14, but 2-tert-butoxycarbonylamino-propionicacid 2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.ESI-MS m/z 413.1 (MH⁺). Compound 27 is shown below.

2-[2-benzyloxy-1-(9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(28)

Compound 28 was prepared using the general procedure described abovewith respect to compound 14, but3-benzyloxy-2-tert-butoxycarbonylaminopropionic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. MS (ES): m/z 504 (M+H), 396, 261. Compound 28 is shown below.

2-(1-(2-amino-9H-purin-6-ylamino)-2-benzyloxy-ethyl-5-methyl-3-phenyl-3H-quinazolin-4-one(29)

Compound 29 was prepared using the general procedure described abovewith respect to compound 14, but3-benzyloxy-2-tert-butoxycarbonylaminopropionic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.MS (ES): m/z 519 (M+H), 411, 261. Compound 29 is shown below.

2-[2-benzyloxy-1-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(30)

Compound 30 was prepared using the general procedure described abovewith respect to compound 14, but3-benzyloxy-2-tert-butoxycarbonylaminopropionic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was substituted for6-bromopurine in step D. MS (ES): m/z 503 (M+H), 395. Compound 30 isshown below.

2-[2-benzyloxy-1-(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(31)

Compound 31 was prepared using the general procedure described abovewith respect to compound 14, but3-benzyloxy-2-tert-butoxycarbonylaminopropionic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-fluoro-6-chloropurine was substituted for 6-bromopurine in step D.MS (ES): m/z 522 (M+H), 414, 261. Compound 31 is shown below.

3-(4-fluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(32)

Compound 32 was prepared using the general procedure described abovewith respect to compound 14, but, with the following changes:4-fluoroaniline was substituted for aniline in step A,2-tert-butoxycarbonylamino-propionic acid 2,5-dioxopyrrolidin-1-yl esterwas substituted for 2-benzyloxycarbonylaminobutyric acid2,5-dioxo-pyrrolidin-1-yl ester in step B, and the alternate procedure(TFA deprotection) was used in step C. ESI-MS m/z 416.1 (MH⁺). Compound32 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(4-fluoro-phenyl)-5-methyl-3H-quinazolin-4-one(33)

Compound 33 was prepared using the general procedure described abovewith respect to compound 14, but 4-fluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was-used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.ESI-MS m/z 431.1 (MH⁺). Compound 33 is shown below.

3-(4-fluoro-phenyl)-2-[1-(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3H-quinazolin-4-one(34)

Compound 34 was prepared using the general procedure described abovewith respect to compound 14, but, 4-fluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-fluoro-6-chloropurine was substituted for 6-bromopurine in step D.ESI-MS m/z 434.1 (MH⁺). Compound 34 is shown below.

3-(4-fluoro-phenyl)-5-methyl-2-[1-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-3H-quinazolin-4-one(35)

Compound 35 was prepared using the general procedure described abovewith respect to compound 14, but 4-fluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was substituted for6-bromopurine in step D. ESI-MS m/z 415.1 (MH⁺). Compound 35 is shownbelow.

5-methyl-3-phenyl-2-[1-(7H-pyrrolo[23-d]pyrimidin-4-ylamino)-ethyl]-3H-quinazolin-4-one (36)

Compound 36 was prepared using the general procedure described abovewith respect to compound 14, but 2-tert-butoxycarbonylamino-propionicacid 2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was substituted for6-bromopurine in step D. ESI-MS m/z 397 (MH⁺). Compound 36 is shownbelow.

3-(3-fluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(37)

Compound 37 was prepared using the general procedure described abovewith respect to compound 14, but 3-fluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ¹HNMR (dmso-d6): 8.30-8.28 (m, 2H); 7.70-7.49 (m, 3H); 7.44-7.30 (m,4H); 4.91-4.88 (m, 1H); 2.72-2.68 (m, 3 h); 1.50-1.48 (m, 3H). Compound37 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-fluoro-phenyl)-5-methyl-3H-quinazolin-4-one(38)

Compound 38 was prepared using the general procedure described abovewith respect to compound 14, but 3-fluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.¹H NMR (dmso-d6): 8.16-8.12 (m, 1H); 7.74-7.69 (m, 1H); 7.62-7.53 (m,2H); 7.46-7.12 (m, 6H); 4.96 (bs, 1H); 2.74-2.67 (m, 3H); 1.47-1.45 (m,3H). Compound 38 is shown below.

3-(3-fluoro-phenyl)-5-methyl-2-[1-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-3H-quinazolin-4-one(39)

Compound 39 was prepared using the general procedure described abovewith respect to compound 14, but 3-fluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was substituted for6-bromopurine in step D. ESI-MS m/z 415.1 (MH⁺). Compound 39 is shownbelow.

5-methyl-3-phenyl-2-[1-(9H-purin-6-yl)-pyrrolidin-2-yl]-3H-quinazolin-4-one(40)

Compound 40 was prepared using the general procedure described abovewith respect to compound 14, but pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-(2,5-dioxo-pyrrolidin-1-yl) ester was substitutedfor 2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl esterin step 8, and the alternate procedure (TFA deprotection) was used instep C. MS (ES): m/z 424 (M+H), 212. Compound 40 is shown below.

2-[2-hydroxy-1-(9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(41)

A suspension of compound 28 (60 mg, 0.097 mmol), Pd(OH)₂ (cat.), andaqueous NazCO₃ (0.5 mL) in ethanol (2.5 mL) was hydrogenated at 45 psifor 14 days. The mixture was filtered to removed solvents, and theresulting filtrate was purified by HPLC to provide the product 41 as awhite solid. MS (ES): m/z 414 (M+H), 396, 261. Compound 41 is shownbelow.

5-methyl-3-phenyl-2-[phenyl-(9H-purin-6-ylamino)-methyl]-3H-quinazolin-4-one(42)

Compound 42 was prepared using the general procedure described abovewith respect to compound 14, but tert-butoxycarbonylamino-phenyl-aceticacid 2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. MS (ES): m/z 460 (M+H), 325. Compound 42 is shown below.

2-[(2-amino-9H-purin-6-ylamino)-phenyl-methyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(43)

Compound 43 was prepared using the general procedure described abovewith respect to compound 14, but tert-butoxycarbonylamino-phenyl-aceticacid 2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.MS (ES): m/z 475 (M+H). Compound 43 is shown below.

2-[(2-fluoro-9H-purin-6-ylamino)-phenyl-methyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(44)

Compound 44 was prepared using the general procedure described abovewith respect to compound 14, but tert-butoxycarbonylamino-phenyl-aceticacid 2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-fluoro-6-chloropurine was substituted for 6-bromopurine in step D.MS (ES): m/z 478 (M+H), 325. Compound 44 is shown below.

5-methyl-3-phenyl-2-[phenyl-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-methyl]-3H-quinazolin-4-one(45)

Compound 45 was prepared using the general procedure described abovewith respect to compound 14, but tert-butoxycarbonylamino-phenyl-aceticacid 2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was substituted for6-bromopurine in step D. MS (ES): m/z 459 (M+H), 230. Compound 45 isshown below.

5-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(46)

Compound 46 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-6-fluoro-benzoic acid wassubstituted for 2-amino-6-methyl-benzoic acid in step A,2-tert-butoxycarbonylamino-propionic acid 2,5-dioxopyrrolidin-1-yl esterwas substituted for 2-benzyloxycarbonylaminobutyric acid2,5-dioxo-pyrrolidin-1-yl ester in step B, and the alternate procedure(TFA deprotection) was used in step C. ESI-MS m/z 402.3 (MH⁺). Compound46 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-fluoro-3-phenyl-3H-quinazolin-4-one(47)

Compound 47 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-6-fluoro-benzoic acid wassubstituted for 2-amino-6-methyl-benzoic acid in step A,2-tert-butoxycarbonylamino-propionic acid 2,5-dioxopyrrolidin-1-yl esterwas substituted for 2-benzyloxycarbonylaminobutyric acid2,5-dioxo-pyrrolidin-1-yl ester in step B, the alternate procedure (TFAdeprotection) was used in step C, and 2-amino-6-bromopurine wassubstituted for 6-bromopurine in step D. ESI-MS m/z 417.2 (MH⁺).Compound 47 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-chloro-3-phenyl-3H-quinazolin-4-one(48)

Compound 48 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-6-chlorobenzoic acid wassubstituted for 2-amino-6-methylbenzoic acid in step A,2-tert-butoxycarbonylamino-propionic acid 2,5-dioxopyrrolidin-1-yl esterwas substituted for 2-benzyloxycarbonylaminobutyric acid2,5-dioxo-pyrrolidin-1-yl ester in step B, the alternate procedure (TFAdeprotection) was used in step C, and 2-amino-6-bromopurine wassubstituted for 6-bromopurine in step D. MS (ES): m/z 433 (M+H), 177.Compound 48 is shown below.

[5-(5-methy-4-oxo-1-phenyl-34-dihydro-quinazolin-2-yl)-5-(9H-purin-6-ylamino)-pentyl]-carbamic acidbenzyl ester (49)

Compound 49 was prepared using the general procedure described abovewith respect to compound 14, but6-benzyloxycarbonylamino-2-tert-butoxycarbonylamino-hexanoic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep 8, and the alternate procedure (TFA deprotection) was used in stepC. ESI-MS m/z 589 (MH⁺). Compound 49 is shown below.

[5-(2-amino-9H-purin-6-ylamino)-5-(5-methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-pentyl]-carbamicacid benzyl ester (50)

Compound 50 was prepared using the general procedure described abovewith respect to compound 14, but6-benzyloxycarbonylamino-2-tert-butoxycarbonylamino-hexanoic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.ESI-MS m/z 604 (MH⁺). Compound 50 is shown below.

[4-(5-methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-4-(9H-purin-6-ylamino)-butyl]-carbamicacid benzyl eater (51)

Compound 51 was prepared using the general procedure described abovewith respect to compound 14, but6-benzyloxycarbonylamino-2-tert-butoxycarbonylamino-pentanoic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ESI-MS m/z 575 (MH⁺). Compound 51 is shown below.

[4-(2-amino-9H-purin-6-ylamino)-4-(5-methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-butyl]-carbamicacid benzyl ester (52)

Compound 52 was prepared using the general procedure described abovewith respect to compound 14, but6-benzyloxycarbonylamino-2-tert-butoxycarbonylamino-pentanoic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.ESI-MS m/z 590 (MH⁺). Compound 52 is shown below.

3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one (53)

Compound 53 was prepared using the general procedure described abovewith respect to compound 14, but anthranilic acid was substituted for2-amino-6-methylbenzoic acid in step A,2-tert-butoxycarbonylamino-propionic acid 2,5-dioxopyrrolidin-1-yl esterwas substituted for 2-benzyloxycarbonylaminobutyric acid2,5-dioxo-pyrrolidin-1-yl ester in step B, and the alternate procedure(TFA deprotection) was used in step C. MS (ES): m/z 384.1 (M+H).Compound 53 is shown below.

2-[5-amino-1-(9H-purin-6-ylamino)-pentyl]-5-methyl-3-phenyl-3H-quinazolin-4-one)(54)

A mixture of compound 49 (28.5 mg) and palladium on carbon (10 mg, 10%Pd) in ethanol (2 mL) was shaken with hydrogen (40 psi) for 48 h. Themixture was filtered through CELITE® and the filtrate was concentratedto afford the product 54. ESI-MS m/z 455 (MH⁺). Compound 54 is shownbelow.

2-[5-amino-1-(2-amino-9H-purin-6-ylamino)-pentyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(55)

Compound 55 was prepared using the general procedure described abovewith respect to compound 54, but compound 50 was substituted forcompound 49. ESI-MS m/z 470 (MH⁺). Compound 55 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-Dimethyl-phenyl)-5-methyl-3H-quinazolin-4-one (56)

Compound 56 was prepared using the general procedure described abovewith respect to compound 14, but 2,6-dimethylaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.ESI-MS m/z 441 (MH⁺). Compound 56 is shown below.

3-(2,6-dimethyl-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(57)

Compound 57 was prepared using the general procedure described abovewith respect to compound 14, but 2,6-dimethylaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ESI-MS m/z 426 (MH⁺). Compound 57 is shown below.

5-morpholin-4-ylmethyl-3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(58)

Compound 58 was prepared using steps B, C, and D of the generalprocedure described above with respect to compound 14, but intermediatecompound 2 was substituted for the product of step A in step B (step Awas thus not needed), 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ESI-MS m/z 483 (MH⁺). Compound 58 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-morpholin-4ylmethyl-3-phenyl-3H-quinazolin-4-one(59)

Compound 59 was prepared using the general procedure described abovewith respect to compound 58, but 2-amino-6-bromopurine was substitutedfor 6-bromopurine in step D. ESI-MS m/z 498 (MH⁺). Compound 59 is shownbelow.

2-[4-amino-1-(2-amino-9H-purin-6-ylamino)-butyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(60)

Compound 60 was prepared using the general procedure described abovewith respect to compound 54, but compound 52 was substituted forcompound 49. ESI-MS m/z 456 (MH⁺). Compound 60 is shown below.

6-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(61)

Compound 61 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-5-fluorobenzoic acid wassubstituted for 2-amino-5-methylbenzoic acid in step A,2-tert-butoxycarbonylamino-propionic acid 2,5-dioxopyrrolidin-1-yl esterwas substituted for 2-benzyloxycarbonylaminobutyric acid2,5-dioxo-pyrrolidin-1-yl ester in step C, and the alternate procedure(TFA deprotection) was used in step C. ESI-MS m/z 402 (MH⁺). Compound 61is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-6-fluoro-3-phenyl-3H-quinazolin-4-one(62)

Compound 62 was prepared using the general procedure described abovewith respect to compound 61, but 2-amino-6-bromopurine was substitutedfor 6-bromopurine in step D. ESI-MS m/z 417 (MH⁺). Compound 62 is shownbelow.

2-[2-tert-butoxy-1-(9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(63)

Compound 63 was prepared using the general procedure described abovewith respect to compound 14, but2-benzyloxycarbonylamino-3-tert-butoxy-propionic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B. MS (ES): m/z 470 (M+H), 396, 261. Compound 63 is shown below.

3-(3-methyl-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(64)

Compound 64 was prepared using the general procedure described abovewith respect to compound 14, but m-toluidine was substituted for anilinein step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ¹H NMR (dmso-d6): 8.41-8.36 (m, 2H); 7.71-7.66 (m, 1H); 7.53-7.51 (m,5H); 4.97 (m, 1H); 2.72 (s, 3H); 2.39 (s, 1.5H); 2.09 (s, 1.5H);1.50-1.48 (m, 3H). Compound 64 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-methyl-phenyl)-5-methyl-3H-quinazolin-4-one(65)

Compound 65 was prepared using the general procedure described abovewith respect to compound 14, but m-toluidine was substituted for anilinein step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.¹H NMR (dmso): 8.94-8.92 (m, 1H); 8.18 (s, 1H); 7.75-7.68 (m, 1H);7.58-7.51 (m, 1H); 5.07-4.96 (m, 1H); 2.79-2.73 (m, 3H); 2.40 (s, 1.5H);1.91 (s, 1.5H); 1.48-1.43 (m, 3H). Compound 65 is shown below.

3-(3-chloro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(66)

Compound 66 was prepared using the general procedure described abovewith respect to compound 14, but 3-chloroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ¹H NMR (dmso-d6): 8.40-8.31 (m, 2H); 7.73-7.66 (m, 1H); 7.55-7.32 (m,6H); 5.04-4.86 (m, 1H); 2.72 (s, 3H); 1.52-1.50 (m, 3H). Compound 66 is,shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-chloro-phenyl)-5-methyl-3H-quinazolin-4-one(67)

Compound 67 was prepared using the general procedure described abovewith respect to compound 66, but 2-amino-6-bromopurine was substitutedfor 6-bromopurine in step D. ESI-MS m/z 447.2 (MH⁺). Compound 67 isshown below.

2-[1-(2-amino-9H-purin-6-ylamino)-2-hydroxy-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(68)

Compound 68 was prepared using the general procedure described abovewith respect to compound 14, but2-benzyloxycarbonylamino-3-tert-butoxy-propionic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep 8, and 2-amino-6-bromopurine was substituted for 6-bromopurine instep D. The obtained2-[1-(2-amino-9H-purin-6-ylamino)-2-tert-butoxy-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-onewas then dissolved in trifluoroacetic acid and allowed to stir atambient temperature for 5 hours. Purification by LC provided the product68 as a white solid. MS (ES): m/z 429 (M+H), 215, 206, 151. Compound 68is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-fluoro-phenyl)-3H-quinazolin-4-one(69)

Compound 69 was prepared using the general procedure described abovewith respect to compound 14, but 3-fluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.ESI-MS m/z 417.1 (MH⁺). Compound 69 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-difluoro-phenyl)-3H-quinazolin-4-one(70)

Compound 70 was prepared using the general procedure described abovewith respect to compound 14, but 2,6-difluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.ESI-MS m/z 435.1 (MH⁺). Compound 70 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-5-fluoro-3-phenyl-3H-quinazolin-4-one(71)

Compound 71 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-5-fluorobenzoic acid wassubstituted for 2-amino-5-methylbenzoic acid in step A, and2-amino-6-bromopurine was substituted for 6-bromopurine in step D.ESI-MS m/z 431 (MH⁺). Compound 71 is shown below.

5-chloro-3-(3-fluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(72)

Compound 72 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-6-chlorobenzoic acid wassubstituted for 2-amino-6-methylbenzoic acid and 3-fluoroaniline wassubstituted for aniline in step A, 2-tert-butoxycarbonylamino-propionicacid 2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ESI-MS m/z 436.1 (MH⁺). Compound 72 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-chloro-3-(3-fluoro-phenyl)-3H-quinazolin-4-one(73)

Compound 73 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-6-chlorobenzoic acid wassubstituted for 2-amino-6-methylbenzoic acid, and 3-fluoroaniline wassubstituted for aniline in step A, 2-tert-butoxycarbonylamino-propionicacid 2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 2-amino-6-bromopurine was substituted for 6-bromopurine in step D.ESI-MS m/z 451.1 (MH⁺). Compound 73 is shown below.

3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-5-trifluoromethyl-3H-quinazolin-4-one(74)

Compound 74 was prepared using the general procedure described abovewith respect to compound 14, but2-amino-N-phenyl-6-trifluoromethyl-benzamide was used in place ofcompound 15 in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used. MS(ES): m/z 452 (M+H). Compound 74 is shown below.

2-amino-N-phenyl-6-trifluoromethyl-benzamide was prepared by addition ofaniline (1.0 eq.) to a suspension of 2-amino-6-(trifluoromethyl)-benzoicacid trihydrate (1.0 g, 4.0 mmol, 1.3 eq.) and polystyrene-carbodiimide(3.6 g, 1.1-1.7 eq.) in THF (40 mL). The reaction was stirred at ambienttemperature for 18 hours, then filtered. The filtrate was concentratedin vacuo and purified by flash chromatography to provide2-amino-N-phenyl-6-trifluoromethyl-benzamide as a yellow solid.

3-(2,6-difluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one(75)

Compound 75 was prepared using the general procedure described abovewith respect to compound 14, but 2,6-difluoroanline was substituted foraniline in step A. ESI-MS m/2 448 (MH⁺). Compound 75 is shown below.

3-(2,6-difluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(76)

Compound 76 was prepared using the general procedure described abovewith respect to compound 14, but 2,6-difluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ESI-MS m/z 434.1 (MH⁺). Compound 76 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-3-(2,6-difluoro-phenyl)-5-methyl-3H-quinazolin-4-one(77)

Compound 77 was prepared using the general procedure described abovewith respect to compound 75, but 2-amino-6-bromopurine was substitutedfor 6-bromopurine in step D. ESI-MS m/z 463 (MH⁺). Compound 77 is shownbelow.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-difluoro-phenyl)-5-methyl-3H-quinazolin-4-one(78)

Compound 78 was prepared using the general procedure described abovewith respect to compound 76, but 2-amino-6-bromopurine was substitutedfor 6-bromopurine in step D. ESI-MS m/z 449 (MH⁺). Compound 78 is shownbelow.

3-(3,5-dichloro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(79)

Compound 79 was prepared using the general procedure described abovewith respect to compound 14, but 3,5-dichloroaniline was substituted foraniline in step 1, and 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ESI-MS m/z 467 (MH⁺). Compound 79 is shown below.

3-(2,6-dichloro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(80)

Compound 80 was prepared using the general procedure described abovewith respect to compound 14, but 2,6-dichloroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ESI-MS m/z 467 (MH⁺). Compound 80 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-dichloro-phenyl)-5-methyl-3H-quinazolin-4-one (81)

Compound 81 was prepared using the general procedure described abovewith respect to compound 80, but 2-amino-6-bromopurine was substitutedfor 6-bromopurine in step D. ESI-MS m/z 482 (MH⁺). Compound 81 is shownbelow.

5-chloro-3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one(82)

Compound 82 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-6-chloro-benzoic acid wassubstituted for 2-amino-6-methyl-benzoic acid in step A,2-tert-butoxycarbonylamino-propionic acid 2,5-dioxopyrrolidin-1-yl esterwas substituted for 2-benzyloxycarbonylaminobutyric acid2,5-dioxo-pyrrolidin-1-yl ester in step B, and the alternate procedure(TFA deprotection) was used in step C. ESI-MS m/z 432 (MH⁺). Compound 82is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-5-chloro-3-phenyl-3H-quinazolin-4-one(83)

Compound 83 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-6-bromopurine was substitutedfor 6-bromopurine in step D. ESI-MS m/z 447 (MH⁺). Compound 83 is shownbelow.

5-methyl-3-phenyl-[1-H-purin-6-ylamino)-butyl]-3H-quinazolin-4-one (84)

Compound 84 was prepared using the general procedure described abovewith respect to compound 14, but 2-benzyloxycarbonylamino-pentanoic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B. MS (ES): m/z 426 (M+H), 213. Compound 84 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-butyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(85)

Compound 85 was prepared using the general procedure described abovewith respect to compound 14, but 2-benzyloxycarbonylamino-pentanoic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and 2-amino-6-bromopurine was substituted for 6-bromopurine instep D. MS (ES): m/z 441 (M+H), 221. Compound 85 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3,5-dichloro-phenyl)-5-methyl-3H-quinazolin-4-one(86)

Compound 86 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-6-bromopurine was substitutedfor 6-bromopurine in step D. ESI-MS m/z 482 (MH⁺). Compound 86 is shownbelow.

5-methyl-3-(3-morpholin-4-ylmethyl-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(87)

Compound 87 was prepared using the general procedure described abovewith respect to compound 14, but intermediate compound 8 was substitutedfor aniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ESI-MS m/z 497 (MH⁺). Compound 87 is shown below.

2-(1-(2-amino-9H-purin-6-ylamino)-ethyl)-5-methyl-3-(3-morpholin-4-ylmethyl-phenyl)-3H-quinazolin-4-one(88)

Compound 88 was prepared using the general procedure described abovewith respect to compound 87, but 2-amino-6-bromopurine was substitutedfor 6-bromopurine in step D. ESI-MS m/z 498 (MH⁺) Compound 88 is shownbelow.

2-[1-(5-bromo-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(89)

Compound 89 was prepared using the general procedure described abovewith respect to compound 14, but 2-tert-butoxycarbonylamino-propionicacid 2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 4-chloro-5-bromo-7H-pyrrolo[2,3-d]pyrimidine (prepared as in J. Med.Chem. 1988, 31, 2086-20′92) was substituted for 6-bromopurine in step D.ESI-MS m/z 411.1. (MH⁺). Compound 89 is shown below.

5-methyl-2-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-3-phenyl-3H-quinazolin-4-one(90)

Compound 90 was prepared using the general procedure described abovewith respect to compound 14, but 2-tert-butoxycarbonylamino-propionicacid 2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (prepared as in J.Med. Chem. 1990, 33, 1984-1992) was substituted for 6-bromopurine instep D. ESI-MS m/z 411.1 (MH⁺). Compound 90 is shown below.

2-[1-(5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(91)

Compound 91 was prepared using the general procedure described abovewith respect to compound 14, but 2-tert-butoxycarbonylamino-propionicacid 2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, the alternate procedure (TFA deprotection) was used in step C,and intermediate compound 1 was substituted for 6-bromopurine. ESI-MSm/z 415.1 (MH⁺). Compound 91 is shown below.

2-[2-hydroxy-1-(9H-purin-6-ylamino)-ethyl]-3-phenyl-3H-quinazolin-4-one(92)

Compound 92 was prepared by adding trifluoroacetic acid to a solution of2-[2-tert-butoxy-1-(9H-purin-6-ylamino)-ethyl]-3-phenyl-3H-quinazolin-4-onein dichloromethane. The reaction was stirred at ambient temperature for18 hours, then concentrated in vacuo. Purification by LC provided theproduct as a white solid. MS (ES): m/z 400 (M+H), 382, 200, 136.Compound 92 is shown below.

2-[2-tert-butoxy-1-(9H-purin-6-ylamino)-ethyl]-3-phenyl-3H-quinazolin-4-onewas prepared using the general procedure described above with respect tocompound 14, but 2-amino-6-chlorobenzoic acid was substituted for2-amino-6-methylbenzoic acid in step A,2-benzyloxycarbonylamino-3-tert-butoxy-propionic acid2,5-dioxo-pyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the conditions used in step C removed both the benzylprotecting group and the A-Ring chloro substituent.

3-(3,5-difluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one(93)

Compound 93 was prepared using the general procedure described abovewith respect to compound 14, but 3,5-difluoroaniline was substituted foraniline in step A. ESI-MS m/z 448 (MH⁺). Compound 93 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-3-(3,5-difluoro-phenyl)-5-methyl-3H-quinazolin-4-one(94)

Compound 94 was prepared using the general procedure described abovewith respect to compound 90, but 2-amino-6-bromopurine was substitutedfor 6-bromopurine in step D. ESI-MS m/z 463 (MH⁺). Compound 94 is shownbelow.

3-(3,5-difluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(95)

Compound 95 was prepared using the general procedure described abovewith respect to compound 14, but anthranilic acid was substituted for2-amino-6-methylbenzoic acid and 3,5-difluoroaniline for aniline in stepA, 2-tert-butoxycarbonylamino-propionic acid 2,5-dioxopyrrolidin-1-ylester was substituted for 2-benzyloxycarbonylaminobutyric acid2,5-dioxo-pyrrolidin-1-yl ester in step B, and the alternate procedure(TFA deprotection) was used in step C. ESI-MS m/z 420 (MH⁺). Compound 95is shown below.

2-[1-(5-bromo-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-3-(3-fluoro-phenyl)-5-methyl-3H-quinazolin-4-one(96)

Compound 96 was prepared using the general procedure described abovewith respect to compound 14, but 3-fluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ESI-MS m/z 494 (MH⁺). Compound 96 is shown below.

3-(3-fluoro-phenyl)-5-methyl-2-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-3H-quinazolin-4-one(97)

Compound 97 was prepared using the general procedure described abovewith respect to compound 14, but 3-fluoroaniline was substituted foraniline in step A, 2-tert-butoxycarbonylamino-propionic acid2,5-dioxopyrrolidin-1-yl ester was substituted for2-benzyloxycarbonylaminobutyric acid 2,5-dioxo-pyrrolidin-1-yl ester instep B, and the alternate procedure (TFA deprotection) was used in stepC. ESI-MS m/z 429 (MH⁺). Compound 97 is shown below.

3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one (98)

Compound 98 was prepared using the general procedure described abovewith respect to compound 14, but anthranilic acid was substituted for2-amino-6-methyl-benzoic acid in step A. MS (ES): m/z 398 (M+H), 199.Compound 98 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3,5-difluoro-phenyl)-3H-quinazolin-4-one(99)

Compound 99 was prepared using the general procedure described abovewith respect to compound 95, but 2-amino-6-bromopurine was substitutedfor 6-bromopurine in step D. ESI-MS m/z 435 (MH⁺). Compound 96 is shownbelow.

2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-3-phenyl-3H-quinazolin-4-one(100)

Compound 100 was prepared using the general procedure described abovewith respect to compound 14, but anthranilic acid was substituted for2-amino-6-methyl-benzoic acid in step A, and 2-amino-6-bromopurine wassubstituted for 6-bromopurine in step D. MS (ES): m/z 413 (M+H), 207.Compound 100 is shown below.

6,7-difluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(101)

Compound 101 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-4,5-difluoro-benzoic acid wassubstituted for 2-amino-6-methyl-benzoic acid in step A,2-tert-butoxycarbonylamino-propionic acid 2,5-dioxopyrrolidin-1-yl esterwas substituted for 2-benzyloxycarbonylaminobutyric acid2,5-dioxo-pyrrolidin-1-yl ester in step B, and the alternate procedure(TFA deprotection) was used in step C. ESI-MS m/z 420 (MH⁺). Compound101 is shown below.

6-fluoro-3-(3-fluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(102)

Compound 102 was prepared using the general procedure described abovewith respect to compound 14, but 2-amino-5-fluoro-benzoic acid wassubstituted for 2-amino-6-methyl-benzoic acid in step A,2-tert-butoxycarbonylamino-propionic acid 2,5-dioxopyrrolidin-1-yl esterwas substituted for 2-benzyloxycarbonylaminobutyric acid2,5-dioxo-pyrrolidin-1-yl ester in step B, and the alternate procedure(TFA deprotection) was used in step C. ESI-MS m/z 420 (MH⁺). Compound102 is shown below.

2-[4-diethylamino-1-(9H-purin-6-ylamino)-butyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(103)

Compound 103 was prepared following steps A-D below.

[4-amino-1-(5-methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-butyl]-carbamicacid tert-butyl ester (104)

Step A: A 10-mL, one-neck, round bottomed flask equipped with a magneticstirrer was purged with nitrogen and charged with 10% palladium oncarbon (30 mg, 50% wet). A solution of[4-benzyloxycarbonylamino-1-(5-methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-butyl]-carbamicacid tert-butyl ester (product from step B of procedure for compound 51)(100 mg, 0.18 mmol) in ethanol (2 mL) and polymethylhydrosiloxane (130mg) were then added sequentially. The reaction mixture was stirred at50° C. for 3 h, and then cooled to room temperature. The mixture wasfiltered through a pad of CELITE® and the filtrate evaporated to drynessSubsequent purification of the resulting crude product by columnchromatography afforded a 62% yield of compound 101 as a white solid. ¹HNMR (CD₃OD) δ 7.55-7.69 (m, 5H), 7.33-7.49 (m, 2H), 7.30 (d, 1H, J=7.3Hz), 4.29 (m, 1H), 2.77 (s, 3H), 2.38-2.47 (m, 2H), 1.72-1.88 (m, 1H),1.57-1.79 (m, 1H), 1.13-1.55 (m, 4H), 1.40 (s, 9H). The reactiondescribed above and compound 104 are shown below.

[4-Diethylamino-1-(5-methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-butyl]-carbamicacid tert-butyl ester (105)

Step B: A 10-mL, one-neck, round bottomed flask equipped with a magneticstirrer was purged with nitrogen, and then charged with the compound 104(100 mg, 0.24 mmol) and 1,2-dichloroethane (2 mL). Acetaldehyde (42 mg,0.85 mmol) and sodium triacetoxyborohydride (400 mg, 1.90 mmol) weresubsequently added. The reaction mixture was stirred for 18 h at ambienttemperature, evaporated to dryness, and the resulting residue wasdissolved in methanol (20 mL). This methanolic solution was treated with10% palladium on carbon (5 mg, 50% wet), stirred for 30 min, evaporatedto dryness, and partitioned between 10% aqueous potassium carbonate (20mL) and methylene chloride (20 mL). The organic layer was separated anddried over sodium sulfate. Filtration of the organic layer followed byconcentration gave the compound 105 as a yellow oil, which was usedwithout any further purification. ¹H NMR (CDCl₃) δ 7.51-7.63 (m, 5H),7.41 (d, 1H, J=7.4 Hz), 7.29 (d, 1H, J=7.15 Hz), 7.22 (d, 1H, J=7.1 Hz),6.10 (d, 1H, J=8.8 Hz), 4.38-4.52 (m, 1H), 2.81 (s, 3H), 2.32-2.50 (m,4H), 2.08-2.27 (m, 2H), 1.47-1.73 (m, 4H), 1.42 (s, 9H, 1.25-1.38 (m,1H), 0.96 (t, 6H, J=7.2 Hz); ESI-MS m/z=479 (MH⁺). The reactiondescribed above and compound 105 are shown below.

2-(1-amino-4-diethylamino-butyl)-5-methyl-3-phenyl-3H-quinazolin-4-one(106)

The alternative deprotection procedure described above with respect tothe preparation of compound 14 (and specifically the preparation ofcompound 17) was used to deprotect compound 105. The reaction andcompound 106 are shown below.

2-[4-Diethylamino-1-(9H-purin-6-ylamino)-butyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(103)

Compound 103 was then prepared following the general procedure providedabove in step D of the procedure for compound 14, but compound 106 wasused as the free amine. ESI-MS m/z 497 (MH⁺). The reaction and compound103 are shown below.

Example 9 Compound Preparation

Compounds in accordance with general formula I (shown above), includingchiral compounds having general formula II (shown above), have beenprepared in accordance with steps A-E of the synthetic scheme shownbelow.

(S)-5-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one(107)

The synthesis of a compound in accordance with formula I is firstexemplified using steps A-E below, which provide a synthetic procedurefor compound 107, the structure of which is shown below.

2-fluoro-6-nitro-N-phenyl-benzamide (108)

Step A: A solution of 2-fluoro-6-nitrobenzoic acid (100 g, 0.54 mol) anddimethylformamide (5 mL) in dichloromethane (600 mL) was treateddropwise with oxalyl chloride (2 M in dichloromethane, 410 mL, 0.8 mol,1.5 eq) over 30 min. After stirring 2 h at room temperature, thereaction was concentrated to an orange syrup with some solids present.The syrup was dissolved in dry dioxane (80 mL) and slowly added to asuspension of aniline (49 mL, 0.54 mol, 1 eq) and sodium bicarbonate (90g, 1.08 mol, 2 eq) in a mixture of dioxane (250 mL) and water (250 mL)at 6° C. The temperature reached 27° C. at the end of the addition.After 30 min, the reaction mixture was treated with water (1.2 L). Theprecipitate was collected by vacuum filtration, washed with water (300mL), air dried in the funnel, and dried in vacuo at 50° C. for 24 h toafford an off-white solid product (139 g, 99%). ¹H NMR (300 MHz,DMSO-d6) δ 10.82 (s, 1H), 8.12 (d, J=7.7 Hz, 1H), 7.91-7.77 (m, 2H),7.64 (d, J=7.7 Hz, 2H), 7.38 (t, J=7.9 Hz, 2H), 7.15 (t, J=7.4 Hz, 1H),ESI-MS m/z 261 (MH⁺). The reaction described above and compound 108 areshown below.

(S)-[1-(2-fluoro-6-nitro-benzoyl)-phenyl-aminocarbonyl]-propyl-carbamicacid tert-butyl ester (109)

Step B: A suspension of compound 108 (0.5 mol) and dimethylformamide (5mL) in thionyl chloride (256 mL, 2.5 mol, 5 eq) was stirred at 85° C.for 5 hours. The reaction mixture was concentrated in vacuo to a brownsyrup. The syrup was dissolved in dichloromethane (200 mL) and wasslowly added to a solution of N—BOC-L-2-aminobutyric acid (112 g, 0.55mol, 1.1 eq) and triethylamine (77 mL, 0.55 mol, 1.1 eq) indichloromethane (600 mL) at 10° C. After stirring at room temperaturefor 3 h, salts were removed by filtration, and the solution was washedwith 100 mL of water, saturated sodium bicarbonate, water, 5% citricacid, and saturated sodium chloride. The organic phase was dried withmagnesium sulfate and concentrated to a red syrup. The syrup wasdissolved in dichloromethane (450 mL) and purified by flashchromatography on a silica gel plug (15×22 cm, 4 L dry silica) elutedwith hexanes/ethyl acetate (10%, 8 L; 15%, 8 L; 20%, 8 L; 25%, 4 L) toyield the compound 109 as an off-white solid (147 g, 66%). ¹H NMR (300MHz, DMSO-d6) δ 8.13 (d, J=8.0 Hz, 1H), 7.84 (t, J=8.6 Hz, 1H),7.78-7.67 (m, 1H), 7.65-7.49 (m, 3H), 7.40-7.28 (m, 2H), 7.19 (d, J=7.5Hz, 1H), 4.05 (broad s, 1H), 1.75-1.30 (m, 2H), 1.34 (s, 9H), 0.93(broad s, 3H). ESI-MS m/z 446.3 (MH⁺). The reaction described above andcompound 109 are shown below.

(S)-[1-(5-fluoro-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-propyl]-carbamicacid tert-butyl ester (110)

Step C: A solution of compound 109 (125 mmol, 1 eq) in acetic acid (500mL) was treated with zinc dust (48.4 g, 740 mmol, 6 eq) added in 3portions, and the reaction mixture was allowed to cool to below 35° C.between additions. After stirring for 2 h at ambient temperature, solidswere filtered off by vacuum filtration and washed with acetic acid (50mL). The filtrate was concentrated in vacuo, dissolved in EtOAc (400mL), washed with water (300 mL), and the water layer was extracted withEtOAc (300 mL). The combined organic layers were washed with water (200mL), sat'd sodium bicarbonate (2×200 mL), sat'd NaCl (100 mL), driedwith MgSO_(q), and concentrated to a syrup. The syrup was dissolved intoluene (200 mL) and purified by flash chromatography on a silica gelplug (13×15 cm, 2 L dry silica) eluted with hexanes/ethyl acetate (10%,4 L; 15%, 4 L; 17.5%, 8 L; 25%, 4 L) to yield compound 110 as anoff-white foamy solid (33.6 g, 69%). ¹H NMR (300 MHz, DMSO-d6) δ 7.83(td, J=8.2, 5.7 Hz, 1H), 7.64-7.48 (m, 5H), 7.39 (broad d, J=7.6 Hz,1H), 7.30 (dd, J=8.3 Hz, 1H), 7.23 (d, J=7.6 Hz, 1H), 4.02-3.90 (m, 1H),1.76-1.66 (m, 1H), 1.62-1.46 (m, 1H), 1.33 (s, 9H), 0.63 (t, J=7.3 Hz,3H). ESI-MS m/z 398.3 (MH⁺). The reaction described above and compound110 are shown below.

(S)-2-(1-amino-propyl)-5-fluoro-3-phenyl-3H-quinazolin-4-one (111)

Step D: A solution of compound 110 (85 mmol) in dichloromethane (60 mL)was treated with trifluoroacetic acid (60 mL). The reaction mixture wasstirred for 1 h, concentrated in vacuo, and partitioned betweendichloromethane (150 mL) and 10% K₂CO₃ (sufficient amount to keep the pHgreater than 10). The aqueous layer was extracted with additionaldichloromethane (100 mL), and the combined organic layers were washedwith water (50 mL) and brine (50 mL). After drying with Mg SO₄, thesolution was concentrated to provide compound 111 as an off-white solid(22 g, 88%). ¹H NMR (300 MHz, CDCl₃) δ 7.73-7.65 (m, 1H); 7.62-7.49 (m,4H), 7.32-7.22 (m, 2H), 7.13-7.06 (m, 1H), 3.42 (dd, J=7.5, 5.2 Hz, 1H),1.87-1.70 (m, 1H), 1.58-1.43 (m, 1H), 0.80 (t, J=7.4 Hz, 3H). ESI-MS m/z298.2 (MH⁺). The reaction described above and compound 111 are shownbelow.

(S)-5-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one(107)

Step E: A suspension of compound 111 (65.6 mmol, 1 eq), 6-bromopurine(14.6 g, 73.4 mmol, 1.1 eq), and DIEA (24.3 mL, 140 mmol, 2 eq) intert-butanol (40 mL) was stirred for 24 h at 80° C. The reaction mixturewas concentrated in vacuo and treated with water to yield a solid crudeproduct that was collected by vacuum filtration, washed with water, andair dried. Half of the obtained solid crude product was dissolved inMeOH (600 mL), concentrated onto silica gel (300 mL dry), and purifiedby flash chromatography (7.5×36 cm, eluted with 10 L of 4% MeOH/CH₂Cl₂)to yield a solid product. The solid product was then dissolved in EtOH(250 mL) and concentrated in vacuo to compound 107 as a light yellowsolid (7.2 g, 50%). ¹H NMR (300 MHz, 80° C., DMSO-d6) δ 12.66 (broad s,1H), 8.11 (s, 1H), 8.02 (broad s, 1H), 7.81-7.73 (m, 1H), 7.60-7.42 (m,6H), 7.25-7.15 (m, 2H), 4.97 (broad s, 1H), 2.02-1.73 (m, 2H), 0.79 (t,J=7.3 Hz, 3H). ESI-MS m/z 416.2 (MH⁺). C, H, N elemental analysis(C₂₂H₁₈N₇OF-EtOH-0.4 H₂O). Chiral purity 99.8:0.2 (S:R) using chiralHPLC (4.6×250 mm Chiralpak ODH column, 20° C., 85:15 hexanes:EtOH, 1mL/min, sample loaded at a concentration of 1 mg/mL in EtOH). Thereaction described above and compound 107 are shown below.

(S)-3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one (112)

Compound 112 was prepared using the general procedure described abovewith respect to compound 107, but 2-nitrobenzoic acid was substitutedfor 2-fluoro-6-nitrobenzoic acid in step A, and N—BOC-L-alanine wassubstituted for N—BOC-L-2-aminobutyric acid in step B. ESI-MS m/z 384.3(MH⁺). Chiral purity 99.5:0.5 (S:R). Compound 112 is shown below.

(S)-6-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(113)

Compound 113 was prepared using the general procedure described abovewith respect to compound 107, but 2-nitro-5-fluorobenzoic acid wassubstituted for 2-fluoro-6-nitrobenzoic acid in step A, andN—BOC-L-alanine was substituted for N—BOC-L-2-aminobutyric acid in stepB. ESI-MS m/z 402.3 (MH⁺). Chiral purity 99.9:0.1 (S:R). Compound 113 isshown below.

(S)-3-(3,5-difluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(114)

Compound 114 was prepared using the general procedure described abovewith respect to compound 107, but 2-nitro-5-methylbenzoic acid wassubstituted for 2-fluoro-6-nitrobenzoic acid and 3,5-difluoroaniline wassubstituted for aniline in step A, and N—BOC-L-alanine was substitutedfor N—BOC-L-2-aminobutyric acid in step B. ESI-MS m/z 434.3 (MH⁺).Compound 114 is shown below.

(S)-5-fluoro-2-(1-(2-fluoro-9H-purin-6-ylamino)-ethyl-3-phenyl-3H-quinazolin-4-one(115)

Compound 115 was prepared using the general procedure described abovewith respect to compound 107, but 2-nitro-6-fluorobenzoic acid wassubstituted for 2-fluoro-6-nitrobenzoic acid in step A, N—BOC-L-alaninewas substituted for N—BOC-L-2-aminobutyric acid in step B, and6-chloro-2-fluoropurine was substituted for 6-bromopurine in step E.ESI-MS m/z 420.3 (MH⁺). Chiral purity 100:0 (S:R). Compound 115 is shownbelow.

(S)-3-(3-fluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(116)

Compound 116 was prepared using the general procedure described abovewith respect to compound 107, but 2-nitrobenzoic acid was substitutedfor 2-fluoro-6-nitrobenzoic acid and 3-fluoroaniline was substituted foraniline in step A, and N—BOC-L-alanine was substituted forN—BOC-L-2-aminobutyric acid in step B. ESI-MS m/z 402.3 (MH⁺). Chiralpurity 96:4 (S:R). Compound 116 is shown below.

(S)-5-chloro-3-(3,5-difluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one(117)

Compound 117 was prepared using the general procedure described abovewith respect to compound 107, but 2-nitro-5-chlorobenzoic acid wassubstituted for 2-fluoro-6-nitrobenzoic acid, and 3,5-difluoroanilinewas substituted for aniline in step A. ESI-MS m/z 468.3 (MH⁺). Chiralpurity 100:0 (S:R). Compound 117 is shown below.

(S)-3-(2,6-difluoro-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(118)

Compound 118 was prepared using an alternative step B relative to thepreparation of compound 107, but the general procedure described abovefor compound 107 was otherwise generally followed for each of steps A,C, and D.

N-(2,6-difluoro-phenyl)-2-methyl-6-nitro-benzamide (118a)

Step A: Compound 118a was prepared following the preparation proceduredescribed above with respect to compound 107, but2-nitro-5-methylbenzoic acid was substituted for 2-fluoro-6-nitrobenzoicacid and 2,6-difluoroaniline was substituted for aniline.

L-(2-[(2,6-difluoro-phenyl)-(2-methyl-6-nitro-benzoyl)-amino]-1-methyl-2-oxo-ethyl)-carbamicacid tert-butyl ester (118b)

Step B: Compound 118b was prepared by treating a solution of compound118a (13.8 g, 47 mmol) in THF (200 mL) dropwise with a solution ofpotassium hexamethyldisilazide (KHMDS) (0.5 M in toluene, 95 mL, 47mmol, 1 eq) at 0° C. and stirring the reaction mixture for 30 min at thesame temperature. The reaction mixture was then treated withL-2-tert-butoxycarbonylamino-propionic acid 2,5-dioxo-pyrrolidin-1-ylester (13.5 g, 47 mmol, 1 eq) and stirred at the same temperature foranadditional 30 min. The reaction mixture was quenched with water (50 mL)and concentrated in vacuo. The residue was dissolved in ethyl acetate(300 mL), and washed with 100 mL of each of the following: (1) water,(2) sat'd sodium bicarbonate, (3) water, (4) 5% citric acid, (5) water,and (6) brine. The organic layer was dried with MgSO₄ and concentratedto a syrup. The crude material was dissolved in dichloromethane (75 mL)and purified by flash chromatography on silica gel (7.5×40 cm), elutedwith 20% EtOAc in hexanes (10 L of 20%, then 6 L of 33%).

Steps C and D were performed as described relative to the preparation ofcompound 107. ESI-MS m/z 434.3 (MH⁺). Chiral purity 100:0 (S:R).Compound 118 is shown below.

(S)-3-(2,6-difluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(119)

Compound 119 was prepared using the general procedure described abovewith respect to compound 118, but 2-nitrobenzoic acid was substitutedfor 2-nitro-5-methylbenzoic acid. ESI-MS m/z 420.3 (MH⁺). Chiral purity100:0 (S:R). Compound 119 is shown below.

5-Methyl-3-phenyl-2-[3,3,3-trifluoro-1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one(120)

Compound 120 was prepared using the general procedure described abovewith respect to compound 107, but 2-methyl-6-nitro-benzoic acid wassubstituted for 2-fluoro-6-nitro-benzoic acid in step A and2-tert-butoxycarbonylamino-4,4,4-trifluoro-butyric acid was substitutedfor 2-tert-butoxycarbonylamino-butyric acid in step B. ESI-MS m/2 466(MH⁺). Compound 120 is shown below.

Example 10 Compound Preparation

Compounds having general formula I (shown above) have been prepared inaccordance with steps A-D of the synthetic scheme entitled “Procedure C”shown below. An alternative synthetic scheme entitled “Scheme D”illustrates additional synthetic routes to compounds having formula I.

Procedure C

The synthesis of compounds in accordance with Procedure C and Scheme Dis first exemplified by the synthetic procedure for3-(3-hydroxy-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one,also referred to as compound 121, the structure of which is shown below.

Compound 121 was prepared following steps A-D below, and using compound122 (below) in step A.

3-(tert-butyl dimethylsilyloxy) phenylamine (122)

A 250 mL, three-neck, round bottomed flask equipped with a magneticstirrer was purged with nitrogen and charged withtert-butyldimethylsilyl chloride (12.5 g, 82.8 mmol), imidazole (7.64 g,112 mmol), and anhydrous DMF (60 mL). 3-aminophenol (10.0 g, 91.7 mmol)was added to the resulting solution. After stirring for 12 h at ambienttemperature, the reaction mixture was poured into water (300 mL). Theresulting suspension was extracted with hexanes (3×300 mL), and theextracts were combined, dried over sodium sulfate, and filtered.Concentration of the filtrate followed by column chromatography gavecompound 122 as a light yellow oil. ¹H NMR (DMSO-d₆) δ (ppm), 6.84 (t,1H, J=7.9 Hz), 6.16 (d, 1H, J=6.7 Hz), 6.10 (m, 1H), 5.97 (d, 1H, J=9.1Hz), 4.98 (s, 2H), 0.95 (s, 9H), 0.16 (s, 6H); m/z=224 (M+H).

2-amino-6-methyl-N-[(3-tert-butyl-dimethyl-silanoxy)-phenyl]-benzamide(123)

Step A: A 5-L, three-neck, round bottomed flask equipped with a gasbubbler, mechanical stirrer and reflux condenser was charged with6-amino-2-methylbenzoic acid (25 g, 16.8 mmol), toluene (300 mL), andthionyl chloride (50 mL). The reaction mixture was refluxed for 1 huntil evolution of gas ceased. The resulting mixture was then cooled andconcentrated under reduced pressure at 50° C. Anhydrous THF (400 mL) andDIEA (90 mL) were added to the resulting residue, followed by compound122 (37 g, 1.0 eq). The resulting reaction mixture was stirred atambient temperature for 2 hours, then quenched by the addition of 20%aqueous potassium carbonate (250 mL). The organic layer was separatedand concentrated to dryness under reduced pressure. Trituration of theresidue with MtBE (70 mL), filtration and drying afforded compound 123.The preparation of compound 123 is generally shown above as step A ofProcedure C.

1-([3-(3-Hydroxy-phenyl)-5-methyl-4-oxo-3,4-dihydro-quinazolin-2-yl]-ethyl)-carbamicacid-tert butyl ester (124)

Step B: A 100-mL, three-neck, round bottomed flask equipped with amagnetic stirrer and a reflux condenser was purged with nitrogen andcharged with compound 123 (10 g, 28 mmol),N-tert-butyloxycarbonylalanine N-hydroxysuccinimide ester (9.60 g, 1.0eq), DMAP (1.90 g), DIEA (6 mL), 4 Å molecular sieves (1.20 g), andanhydrous toluene (100 mL). The resulting mixture was heated in an oilbath at 80° C. for 24 h. 1-Hydroxybenzotriazole (3.78 g) was added tothe reaction, and heating continued for an additional 48 h. Aftercooling, toluene (10 mL) and CELITE® (0.70 g) were added to the warmreaction mixture, followed by filtration and evaporation of the filtrateto give a brown residue. The residue was dissolved in CH2Cl2 (15 mL) andtreated with a solution of tetrabutylammonium fluoride (5.12 g) in MeOH(15 mL). After stirring for 1 h at ambient temperature, the reactionmixture was evaporated to dryness under reduced pressure, and theresidue purified by column chromatography to give 81% yield of compound124 as an off-white solid. The preparation of compound 124 is generallyshown above as step B of Procedure C.

2-(1-amino-ethyl)-3-(3-hydroxy-phenyl)-5-methyl-3H-quinazolin-4-one(125)

Step C1: A 100-mL, three-neck, round bottomed flask equipped with amagnetic stirrer was charged with compound 124 (4.50 g, 11.18 mmol) inCH2Cl2 (15 mL) and TFA (15 mL). After stirring for 1 hr at roomtemperature, the mixture was concentrated under reduced pressure toafford compound 125, which was used as is in the following step.

3-(3-hydroxy-phenyl)-5-methyl-2-(1-[9-(2-trimethylsilanyl-ethoxymethyl)-9H-purin-6-ylamino]-ethyl)-3H-quinazolin-4-one(126)

Step C2: A nitrogen purged, 50-mL one-neck round bottomed flask equippedwith a magnetic stirrer and reflux condenser was charged with compound125 (2.4 g, 8.16 mmol), intermediate compound 10 (2.70 g, 1.0 eq),n-butanol (20 mL), and DIEA (4.2 mL). The mixture was heated at 100° C.for 4 h then cooled to room temperature. Concentration of the reactionmixture under high vacuum-followed by column chromatography gavecompound as a white solid. The preparation of compound 126 is generallyshown above as step C of Procedure C.

3-(3-hydroxy-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(121)

Step D: A 100-mL, one-neck, round bottomed flask equipped with amagnetic stirrer was charged with compound 126 (294 mg, 0.5 mmol), MeOH(15 mL) and 4N hydrochloric acid (15 mL), and the reaction mixture washeated for 5 h at 40° C. Evaporation of the methanol under reducedpressure followed by basification to pH 10 with 10% aqueous potassiumcarbonate gave a white precipitate. The precipitate was filtered, washedwith water and dried under vacuum overnight at ambient temperature toafford compound 121 as a white solid. m/z=414 (M+H). The preparation ofcompound 121 is generally shown above as step D of Procedure C.

3-(3-methoxy-phenyl)-5-methyl-2-(1-[9-(2-trimethylsilanyl-ethoxymethyl)-9H-purin-6-ylamino]-ethyl)-3H-quinazolin-4-one(127)

A 100-mL, one-neck, round bottomed flask equipped with a magneticstirrer was charged with compound 126 (370 mg, 0.68 mmol), potassiumcarbonate (235 mg, 1.70 mmol), and DMF (4 mL). The resulting mixture wasstirred for 5 min, and methyl iodide (435 mg, 3.10 mmol) was then added.After stirring for a further 1 h at ambient temperature, the reactionmixture was evaporated to dryness under reduced pressure. The residuewas purified by column chromatography to give an 82% yield of compound127 as a light yellow oil. ¹H NMR (CD₃OD) δ (ppm) 8.20 (m, 2H),6.99-7.66 (m, 7H), 5.58 (s, 2H), 5.15 (bs, 1H), 3.62 (dt, 2H, J=1.8, 8.0Hz), 3.32 (s, 3H), 2.78 (s, 3H), 1.55 (m, 3H), 0.88 (m, 2H), −0.07 (s9H); m/z=558 (M+H).

3-(3-methoxy-phenyl)-5-methyl-2-(1-[9H-purin-6-ylamino]-ethyl)-3H-quinazolin-4-one(128)

Compound 127 was reacted in accordance with the procedure describedabove for compound 121 (step D) to provide compound 128. m/z=428 (M+H).The structure of compound 128 is shown below.

3-[3-(2-dimethylamino-ethoxy)-phenyl]-5-methyl-2-(1-[9H-purin-6-ylamino]-ethyl)-3H-quinazolin-4-one(129)

Compound 126 (300 mg, 0.54 mmol) was treated with 2-chloroethyldimethylamine hydrochloride salt, at 90° C. for 17 hrs, using theprocedure described above for compound 127. The resulting compound wasthen treated with 4N HCl, in MeOH, using the procedure described forcompound 121 (step D). Compound 129 was obtained. m/z=485 (M+H). Thestructure of compound 129 is shown below.

3-(3-cyclopropylmethoxy-phenyl)-5-methyl-2-(1-[9H-purin-6-ylamino]-ethyl)-3H-quinazolin-4-one(130)

Compound 126 (300 mgs, 0.54 mmol) was treated with bromomethylcyclopropane using the procedure outlined for compound 127, at roomtemperature for 24 hrs. This intermediate was treated with 4N HClaccording to the procedure described for compound 121 (step D). m/z=468(M+H). The structure of compound 130 is shown below.

5-methyl-3-(3-prop-2-ynyloxy-phenyl)-2-(1-[9H-purin-6-ylamino]-ethyl)-3H-quinazolin-4-one(131)

Compound 126 (300 mgs, 0.54 mmol) was treated with propargyl bromide atroom temperature for 24 hrs using the procedure described above forcompound 127. This intermediate was then treated with 4N HCl accordingto the procedure described for compound 121 (step D). m/z=467 (M+H). Thestructure of compound 131 is shown below.

2-{1-[2-amino-9H-purin-6-ylamino]ethyl}-3-(3-hydroxyphenyl)-5-methyl-3H-quinazolin-4-one(132)

Compound 132 was prepared according to the procedures set forth in stepsA and B below.

2-{1-[2-ditert-butyloxycarbonylamino-9-(2-trimethylsilylethoxymethyl)-9H-purin-6-ylamino]ethyl}-3-(3-hydroxyphenyl)-5-methyl-3H-quinazolin-4-one(133)

Step A: A nitrogen purged, 50-mL one-neck round bottomed flask equippedwith a magnetic stirrer and reflux condenser was charged with compound125 (3.02 g, 10.2 mmol), intermediate compound 12 (5.56 g, 1.0 eq),n-butanol (20 mL), and DIPEA (6.0 mL). The mixture was heated at 100° C.for 1 h, and then cooled to room temperature. Concentration of thereaction mixture under high vacuum followed by column chromatographygave compound 133 as a white solid.

2-(1-[2-amino-9H-purin-6-ylamino]ethyl)-3-(3-hydroxyphenyl)-5-methyl-3H-quinazolin-4-one(132)

Step B: Compound 133 was dissolved in MeOH (3 mL), treated with 4N HCl(3 mL) and heated at 40° C. for 6 h. The reaction mixture was thenconcentrated to approximately half the volume and partitioned betweenwater (5 mL) and ethyl acetate (10 mL). The aqueous layer was separated,basified with potassium carbonate to pH 10 and filtered. After washingthe filter cake with water (5 mL) and drying under vacuum, compound 132was obtained as a white solid. m/z=429 (M+H). The structure of compound132 is shown below.

2-{1-[2-amino-9H-purin-6-ylamino]ethyl}-3-(3-methoxyphenyl)-5-methyl-3H-quinazolin-4-one(134)

Compound 133 (300 mgs, 0.39 mmol) was treated with methyl iodide usingthe procedure described above for compound 127. This intermediate wasthen treated with 4N HCl in methanol according to the proceduredescribed above for compound 132 (step B). m/z=443 (M+H). The structureof compound 134 is shown below.

2-{1-[2-amino-9H-purin-6-ylamino]ethyl}-3-(3-cyclopropylmethoxy-phenyl)-5-methyl-3H-quinazolin-4-one(135)

Compound 133 (231 mgs, 0.30 mmol) was treated with cyclopropyl methylbromide using the procedure described above for compound 127. Thegenerated intermediate was then treated with 4N HCl in MeOH according tothe procedure described above for compound 132 (step B). m/z=483 (M+H).The structure of compound 135 is shown below.

2-(1-[2-amino-9H-purin-6-ylamino]ethyl)-5-methyl-3-(3-prop-2-ynyloxy-phenyl)-3H-quinazolin-4-one(136)

Compound 133 (231 mgs, 0.30 mmol) was treated with propargyl bromideusing the procedure described above for compound 127. The generatedintermediate was then treated with 4N HC in MeOH according to theprocedure described above for compound 132 (step B). m/z=467 (M+H). Thestructure of compound 136 is shown below.

3-(3-ethynyl-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(137)

Compound 137 was prepared according to the procedures set forth in stepsA-C below.

Trifluoromethane sulfonic acid3-(5-methyl-4-oxo-2-(1-[9-(2-trimethylsilanyl-ethoxymethyl)-9H-purin-6-ylamino]-ethyl)-4H-quinazolin-3-yl)-phenylester (138)

Step A: A 50-mL, three-neck, round bottomed flask equipped with amagnetic stirrer was purged with nitrogen and charged with compound 126(500 mg, 0.92 mmol), triethylamine (218 mg, 2.16 mmol), anhydrousmethylene chloride (10 mL) and N-phenyltrifluoromethanesulfonimide (496mg, 1.39 mmol). After stirring for 2 h at ambient temperature, thereaction mixture was partitioned between methylene chloride (50 mL) and10% aqueous potassium carbonate (50 mL). The organic phase wasseparated, dried over sodium sulfate and filtered. Concentration of thefiltrate followed by column chromatography gave a 77% yield of compound138 as an off-white solid. ¹H NMR (DMSO-d₆) δ (ppm) 8.32 (bs, 1H),7.48-8.18 (m, 7H), 7.30 (d, 1H, J=8.0 Hz), 5.51 (s, 2H), 4.75-4.85 (m,1H), 3.55 (t, 2H, J=8.0 Hz), 2.72 (s, 3H), 1.46 (d, 3H, J=6.6 Hz), 0.83(dt, 2H, J=1.6, 8.1 Hz), −0.09 (s, 9H).

5-Methyl-2-(1-[9-(2-trimethylsilylethoxymethyl)-9H-purin-6-ylamino]ethyl)-3-(3-trimethylsilylethynylphenyl)-3H-quinazolin-4-one(139)

Step B: A 5-mL reaction vial equipped with a magnetic stirrer was purgedwith nitrogen and charged with compound 138 (220 mg, 0.33 mmol),dichlorobis(triphenylphosphine)palladium(II) (27.1 mg, 0.039 mmol), andanhydrous DMF (1 mL). Triethylamine (146 mg, 1.44 mmol) and(trimethylsilyl)acetylene (102 mg, 1.04 mmol) were added, and thereaction mixture was stirred for 10 h at 90° C. and an additional 8 h at100° C. Evaporation of the reaction mixture to dryness followed bycolumn chromatography purification afforded 63% yield of compound 139 asan off-white solid. ¹H NMR (CD₃OΔ) δ (ppm) 7.43-8.33 (m, 8H), 7.30 (d,1H, J=6.6 Hz), 5.64 (s, 2H), 5.14 (bs, 1H), 3.68 (t, 2H, J=8:0 Hz), 2.81(s, 3H), 1.59-1.64 (m, 3H), 0.94 (m, 2H), 0.32 (s, 9H), −0.09 (s, 9H).

3-(3-ethynyl-phenyl)-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(137)

Step C: Compound 139 (113 mgs, 0.18 mmol) was treated with 4N HCl inMeOH according to the procedure described for compound 121 (step D).This afforded compound 137. m/z=422 (M+H). The structure of compound 137is shown below.

3-{5-methyl-4-oxo-2-[1-(9H-purin-6-ylamino)-ethyl]-4H-quinazolin-3-yl}-benzonitrile(140)

Compound 140 was prepared according to the procedures set forth in stepsA and B below.

3-(5-methyl-4-oxo-2-{1-[9-(2-trimethylsilylethoxymethyl)-9H-purin-6-ylamino]ethyl}-4H-quinazolin-3-yl)benzonitrile(141)

Step A: A 5-mL reaction vial equipped with a magnetic stirrer wascharged with compound 138 (200 mg, 0.300 mmol),tetrakis(triphenylphosphine)palladium (34.0 mg, 0.029 mmol), zinccyanide (70 mg, 0.60 mmol) and anhydrous DMF (1 mL). The vial was purgedwith nitrogen, heated to 120° C. for 3.5 h then cooled to ambienttemperature and poured into a saturated aqueous sodium bicarbonatesolution (25 mL). The resulting suspension was extracted with methylenechloride (3×20 mL), and the organic extracts were combined, dried oversodium sulfate and filtered. Concentration of the filtrate followed bypurification by column chromatography gave a 66% yield of compound 141as a white solid. ¹H NMR (CDCl₃) δ (ppm) 8.30 (d, 1H, J=6.9 Hz),7.58-8.02 (m, 7H), 7.30 (d, 1H, J=7.0 Hz), 5.60 (s, 2H), 5.07 (bs, 1H),3.65 (m, 2H, 2.84 (s, 3H), 1.58 (d, 3H, J=6.7 Hz), 0.96 (m, 2H), 0.02(s, 9H).

3-(5-Methyl-4-oxo-2-[1-(9H-purin-6-ylamino)-ethyl]-4H-quinazolin-3-yl)-benzonitrile(140)

Step B: Compound 141 was treated with 4N HCL in MeOH for 1 hour usingthe procedure described for compound 121 (step B) to provide compound140. m/z=423 (M+H). The structure of compound 140 is shown below.

3-{5-Methyl-4-oxo-2-(1-[9H-purin-6-ylamino)-ethyl]-4H-quinazolin-3-yl}-benzamide(142)

Compound 142 was prepared according to the procedures set forth in stepsA and B below.

3-(5-Methyl-4-oxo-2-(1-[9-(2-trimethylsilylethoxymethyl)-9H-purin-6-ylamino]ethyl)-4H-quinazolin-3-yl)benzamide(143)

Step A: A 100-mL, three-neck, round bottomed flask equipped with amagnetic stirrer and a reflux condenser was purged with nitrogen andcharged with compound 141 (219 mg, 0.40 mmol) and anhydrous methylenechloride (15 mL). N,N-Diethylhydroxylamine (146 mg, 1.64 mmol) was addedto the resulting solution, and the reaction mixture was heated for 16 hat 50° C., cooled to ambient temperature, and then evaporated to drynessunder reduced pressure. The resulting residue was purified by columnchromatography to give a 97% yield of compound 143 as a white solid.m.p. 195-197° C.; ¹H NMR (CDCl₃) δ (ppm) 8.34 (d, 1H, J=10.9 Hz),7.59-8.06 (m, 7H), 7.28 (m, 1H′), 6.95 (bs, 1H), 6.00 (bs, 2H), 5.60 (s,2H), 5.28 (bs, 1H), 3.62 (t, 2H, J=8.4 Hz), 2.85 (s, 3H), 1.53 (dd, 3H,J=6.7, 10.8 Hz), 0.96 (t, 2H, J=8.3 Hz), 0.01 (s, 9H); m/z=571 (M+H).

3-(5-Methyl-4-oxo-2-{1-[9H-purin-6-ylamino)-ethyl]-4H-quinazolin-3-yl}-benzamide(142)

Step B: Compound 143 was treated with 4N HCL in MeOH for 1.5 hours usingthe procedure described for compound 121 (step D) to provide compound142. m/z=441 (M+H). The structure of compound 142 is shown below.

3-(3-acetyl-phenyl)-5-methyl-2-(1-[9H-purin-6-ylamino]-ethyl)-3H-quinazolin-4-one(144)

Compound 139 was treated with 4N HCl in MeOH at 70° C. for 16 hours inaccordance with the procedure described for compound 121 (step D). Thisreaction afforded compound 144, the structure of which is shown below.m/z=440 (M+H)

2-(3-(5-methyl-4-oxo-2-{1-[9H-purin-6-ylamino]-ethyl}-4H-quinazolin-3-yl-phenoxyacetamide (145)

Compound 126 (300 mgs, 0.54 mmol) was treated with 2 bromo acetamideusing the procedure outlined above for compound 127. The reaction wasunder reflux for 24 hrs in CH3CN. This intermediate was treated with 4NHCl in MeOH for 1 hour, following the procedure described for compound121 (step D), to provide compound 145. m/z=471 (M+H). The structure ofcompound 145 is shown below.

5-methyl-2-(1-[9H-purin-6-ylamino]-ethyl)-3-[3-(tetrahydropuran-4-yloxy)-phenyl]-3H-quinazolin-4-one(146)

A 25-mL, three-neck, round bottomed flask equipped with a magneticstirrer was purged with nitrogen and charged with compound 126 (270 mgs,0.5 mmol), tetrahydro pyran-4-ol (60 uL), triphenylphosphine (560 mgs),THF (5 mL), and diethyl azodicarboxylate (340 uL). After stirring for 16h at ambient temperature, the reaction mixture was evaporated todryness, and the residue was dissolved in methanol (3 mL), treated with4N hydrochloric acid (3 mL), and heated at 40° C. for 6 h. The reactionmixture was then concentrated to approximately half the volume andpartitioned between water (5 mL) and ethyl acetate (10 mL). The aqueouslayer was separated, basified with potassium carbonate to pH 10 andfiltered. After washing the filter cake with water (5 mL) and dryingunder vacuum, compound 146 was obtained. m/z=498 (M+H). The structure ofcompound 146 is shown below.

3-[3-(2-methoxy-ethoxy)-phenyl]-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(147)

Compound 126 (300 mgs, 0.54 mmol) was treated with toluene 4-sulfonicacid 2-methoxy ethyl ester at 50 C. for 42 hrs using the proceduredescribed above for compound 127. The generated intermediate was thentreated with 4N HCl in MeOH, using the procedure described for compound121 (step D). m/z=487 (M+H):

6-fluoro-2-[1-(9H-purin-6-ylamino)ethyl]-3-[3-(tetrahydro-pyran-4-yloxy)-phenyl]-3H-quinazolin-4-one(148)

Compound 148 was prepared according to the procedures set forth in stepsA and B below.

6-fluoro-3-(3-hydroxy-phenyl)-2-{1-[9-(2-trimethylsilanyl-ethoxymethyl)-9H-purin-6ylamino-ethyl}-3H-quinazolin-4-one(149)

Step A: Compound 149 was obtained from 6-amino 3-fluoro benzoic acidusing the procedures described above for compounds 123, 124, 125, and126.

6-fluoro-2-[1-(9H-purin-6-ylamino)ethyl]-3-[3-(tetrahydro-pyran-4-yloxy)-phenyl]-3H-quinazolin-4-one (148)

Step B: Compound 148 was obtained from compound 149 using the proceduredescribed for compound 146. m/z=502 (M+H). The structure of compound 148is shown below.

3-[3-(3-dimethylamino-propoxy)-phenyl]-5-methyl-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(150)

Compound 150 was obtained following the general procedure described forcompound 146, but 3-dimethylamino-1-propanol was used in place oftetrahydropyran-4-ol. m/z=499 (M+H). The structure of compound 150 isshown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-ethynyl-phenyl)-5-methyl-3H-quinazolin-4-one(151)

Compound 151 was prepared according to the procedures set forth in stepsA and B below.

Trifluoromethane sulfonic acid 3-(2-[1-(2-ditert-butyloxycarbonylamino-9-(2-trimethylsilylethoxymethyl)-purin-6ylamino)-ethyl]-5-methyl-4-oxo-4H-quinazolin-3-yl)-phenylester (152)

Step A: Compound 152 was obtained from compound 133, which was reactedin accordance with the procedure described for compound 138.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-ethynyl-phenyl)-5-methyl-3H-quinazolin-4-one(151)

Step B: Compound 151 was obtained from compound 152, which was reactedin accordance with the procedures for compounds 139 and 137 (step C).m/z=437 (M+H). The structure of compound 151 is shown below.

3-2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-4-oxo-4H-quinazolin-3-yl)-benzonitrile(153)

Compound 153 was obtained from compound 152, which was reacted inaccordance with the procedure for compounds 141 and 140 (step D)described above. m/z=438 (M+H). The structure of compound 153 is shownbelow.

3-(2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-4-oxo-4H-quinazolin-3-yl)-benzamide(154)

Compound 154 was obtained by first reacting compound 152 in accordancewith the procedure for compound 141. This reaction product was thenfurther reacted in accordance with the procedure for compounds 143 and142 (step B). m/z=456 (M+H). The structure of compound 154 is shownbelow.

3-(2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-4-oxo-4H-quinazolin-3-yl-benzamide(155)

Compound 155 was obtained by first reacting compound 151 in accordancewith the procedure described for compound 139. This reaction product wastreated according to the procedure described for compound 144. m/z=455(M+H). The structure of compound 155 is shown below.

5-methyl-3-(3-morpholin-4-yl-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(156)

Compound 156 was prepared according to the procedures set forth in stepsA and B below.

5-methyl-3-(3-morpholino-4-yl-phenyl)-2-(1-[9-(2-trimethylsilanylethoxymethyl)-9H-purin-6-ylamino]-ethyl)-3H-quinazolin-4-one(157)

Step A: A 3-mL reaction vial was charged with compound 138 (96.1 mg,0.142 mmol), palladium(II) acetate (3.20 mg, 0.014 mmol), cesiumcarbonate (84.2 mg, 0.258 mmol) and (+)-BINAP (i.e.,2,2′-bis(diphenylphosphino)-1,1′-binaphthyl) (13.8 mg, 0.022 mmol). Thevial was then flushed with nitrogen for 10 min. Toluene (0.3 mL) andmorpholine (18 μL) were then added, and the solution was heated at 100°C. for 6 h. Subsequently, the solution was diluted with dichloromethane(5 mL), filtered, and the filtrate was concentrated under reducedpressure. Preparative HPLC of the residue provided compound 157 as ayellow oil. ¹H NMR (CH₃OD) δ (ppm) 8.24-8.30 (m, 2H), 7.48-7.72 (m, 3H),7.33 (m, 2H), 6.94-7.13 (m, 3H), 5.56 and 5.64 (two s, CH₂ rotamer ratio1:10), 5.15-5.30 (m, 1H), 3.90 (m, 2H), 3.76 (m, 2H), 3.67 (m, 2H), 3.28(m, 1H), 2.97-3.15 (m, 3H), 2.84 (s, 3H), 1.62 (m, 3H), 0.96 (m, 2H),−0.03 (s, 9H).

5-methyl-3-(3-morpholin-4-yl-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(156)

Step B: Compound 156 was prepared by reacting compound 157 in accordancewith the procedure for the preparation of compound 137 (final step C).m/z=483 (M+H). The structure of compound 156 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-3-(3-morpholin-4-yl-phenyl)-3H-quinazolin-4-one(158)

Compound 158 was prepared in accordance with the procedure described forcompound 129, but compound 133 was used in place of compound 126.m/z=498 (M+H). The structure of compound 158 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-[3-(2-methoxy-ethoxy)-phenyl]-5-methyl-3H-quinazolin-4-one(159)

Compound 159 was prepared by reacting compound 133 in accordance withthe procedure for the preparation of compound 147. m/z=487 (M+H). Thestructure of compound 159 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-3-[3-(2-dimethylamino-ethoxy)-phenyl]-5-methyl-3H-quinazolin-4-one(160)

Compound 160 was prepared by reacting compound 133 in accordance withthe procedure for the preparation of compound 146. m/z=500 (M+H). Thestructure of compound 160 is shown below.

Example 11 Compound Preparation

Compounds having general formula I (shown above) have been prepared inaccordance with steps A-D of the synthetic scheme entitled “Procedure E”shown below. Procedure E provides an additional alternative method ofpreparing compounds with a variety of side chains appended to the linkerbetween the quinazolinone and purine rings of the inventive compounds.Although a propargyl functional group is exemplified, the methodillustrated in Procedure E is applicable to many known functionalgroups.

Procedure E

2-[1-(2-amino-9H-purin-6-ylamino)-but-3-ynyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(161)

Compound 161 was prepared according to the procedures set forth in stepsA-D below.

2-[(Benzhydrylidene-amino)-methyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(162)

Step A: A 100-mL, one-neck, round bottomed flask equipped with amagnetic stirrer and reflux condenser was charged with2-aminomethyl-5-methyl-3-phenyl-3H-quinazolin-4-one (2.91 g, 11.0 mmol),benzhydrylideneamine (2.39 g, 13.2 mmol), and 1,2-dichloroethane (15mL). The reaction mixture was heated at reflux for 4 h under nitrogenatmosphere and then cooled to ambient temperature. Concentration underreduced pressure followed by purification by column chromatographyafforded the compound 162 as an orange solid. m.p. 49° C. (dec); ¹H NMR(DMSO-d₆) δ (ppm) 7.22-7.78 (m, 17H), 6.74 (m, 1H), 4.17 (s, 2H), 2.74(s, 3H); m/z=430 (M+H). The reaction described above and compound 162are shown below.

2-[1-(Benzhydrylidene-amino)-but-3-ynyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(163)

Step B: A 25-mL, one-neck, round bottomed flask equipped with a magneticstirrer was purged with nitrogen and charged with compound 162 (500 mg,1.20 mmol) and anhydrous THE (4 mL). A 1M solution of potassiumtert-butoxide in THF (1.40 mL, 1.40 mmol) was added in one portion.After stirring for 20 min at ambient temperature, an 80% solution ofpropargyl bromide in toluene (210 μL, 1.89 mmol) was added, and thereaction stirred for an additional 15 min at ambient temperature.Saturated aqueous sodium bicarbonate (5 mL) was then added, the layersseparated, and the aqueous layer extracted with ethyl acetate (2×10 mL).The combined organic extracts were dried over sodium sulfate, filtered,and concentrated under reduced pressure. Purification of the residue bycolumn chromatography afforded the product as a yellow solid. ¹H NMR(CD₃OD) δ 7.72 (m, 2H), 7.24-7.67 (m, 3H), 6.77 (m, 3H), 4.61 (t, 1H,J=7.1 Hz), 3.01-3.11 (m, 1H), 2.76 (s, 3H), 2.57 (m, 1H), 2.23 (t, 1H,J=2.5 Hz). The reaction described above and compound 163 are shownbelow.

2-(1-amino-but-3-ynyl)-5-methyl-3-phenyl-3H-quinazolin-4-one (164)

Step C: A 50-mL, one-neck, round bottomed flask equipped with a magneticstirrer was charged with compound 163 (193 mg, 0.41 mmol) and diethylether (5 mL). A 2N solution of hydrochloric acid (5 mL) was added in oneportion. After stirring for 1.5 h at ambient temperature, sodiumchloride (750 mg, 12.8 mmol) was added to the reaction mixture, andstirring was continued for an additional 10 min. The resultingprecipitate was filtered, washed sequentially with diethyl ether (0.5mL), 2N hydrochloric acid (1 mL), and MtBE (2×1 mL). Drying under vacuumat 45° C. for 2 h afforded the product as a pink solid. ¹H NMR (DMSO-d₆)δ 8.82 (s, 3H), 7.79 (t, 1H, J=7.7 Hz), 7.41-7.66 (m, 6H), 7.42 (d, 1H,J=7.3 Hz), 3.91 9 s, 1H), 3.11 (m, 1H), 2.87 (m, 18), 2.75 (s, 3H),2.54-2.67 (m, 1H). The reaction described above and compound 164 areshown below.

5-methyl-3-phenyl-2-[1-(9H-purin-6-ylamino)-but-3-ynyl]-3H-quinazolin-4-one(161)

Step D: Compound 161 was prepared by reacting compound 164 preparedfollowing the procedure for the preparation of compound 14 (step D)using three equivalents of diisopropylethylamine instead of one. ESI-MSm/z=422 (MH⁺). The reaction described above and compound 161 are shownbelow.

2-[1-(2-amino-9H-purin-6-ylamino)-but-3-ynyl]-5-methyl-3-phenyl-3H-quinazolin-4-one(165)

Compound 165 was prepared following the general procedure describedabove for the preparation of compound 161, but 2-amino-6-bromopurine wassubstituted for 6-bromopurine in step D. ESI-MS m/z=437 (MH⁺).

Example 12 Compound Preparation

Compounds having general formula I (shown above) have been prepared inaccordance with steps A-E of the synthetic scheme entitled “Procedure K”shown below. Procedure K provides an additional alternative method ofpreparing such compounds via an oxazine intermediate (step C).

Procedure K

5-chloro-3-(3,5-difluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(166)

Compound 166 was prepared according to the procedures set forth in stepsA-E below.

2-amino-6-chloro-N-(3,5-difluoro-phenyl)-benzamide (167)

Step A: Compound 167 was prepared following the procedure following theprocedure for the preparation of compound 15 (step A), but2-amino-6-chlorobenzoic acid was substituted for 2-amino-6-methylbenzoicacid and 3,5-difluoroaniline was substituted for aniline. The reactiondescribed above and compound 167 are shown below.

(1-[3-chloro-2-(3,5-difluoro-phenylcarbamoyl)-phenylcarbamoyl]-ethyl)-carbamicacid tert-butyl ester (168)

Step B: A 100-mL, one-neck, round bottomed flask equipped with amagnetic stirrer and reflux condenser was charged with the compound 167(10.6 mmol), N-tert-butyloxycarbonyl-D,L-alanine N-hydroxysuccinimideester (3.64 g, 12.7 mmol), 4-N,N-dimethylaminopyridine (710 mg, 5.02mmol) and 4 Å molecular sieves (3.00 g). The flask was purged withnitrogen, and anhydrous toluene (15 mL) and N,N-diisopropylethylamine(1.64 g, 2.22 mmol) were added. The reaction mixture was heated at 90°C. for 7 h, and the resulting suspension filtered hot. Concentration ofthe filtrate under reduced pressure afforded a light brown solid, whichwas purified by column chromatography (silica gel, EtOAc/hexanes). Thisafforded an 86% yield of compound 168 as a white solid. m.p. 194-196° C.(dec.); ¹H NMR (DMSO-d₆) δ (ppm) 10.96 (s, 1H), 9.46 (s, 1H), 7.84 (d,1H, J=7.8 Hz), 7.51-7.36 (m, 4H), 7.19 (d, 1H, J=6.8 Hz), 6.99 (t, 1H,J=9.3 Hz), 4.10 (t, 1H, J=7.0 Hz), 1.31 (s, 9H), 1.16 (d, 3H, J=6.9 Hz);m/z=454 (M+H). The reaction described above and compound 168 are shownbelow.

(1-[5-chloro-4-(3,5-difluoro-phenylimino)-4H-benzo[d][1,3]oxazin-2-yl]-ethyl)-carbamicacid tert-butyl ester (169)

Step C: A 100-mL, three-neck, round bottomed flask equipped with amagnetic stirrer and thermometer was purged with nitrogen and chargedwith compound 168 (3.30 mmol), anhydrous methylene chloride (25 mL),N,N-diisopropylethylamine (4.60 g, 35.7 mmol) and triphenylphosphine(3.98 g, 15.2 mmol). The reaction mixture was then cooled to 0-5° C. inan ice/water bath. Iodine (3.61 g, 14.2 mmol) was then addedportion-wise to the reaction mixture over 1 h. Once the addition wascomplete, the cooling bath was removed, and the mixture was stirred atroom temperature for an additional 30 min. The reaction was thenquenched with 10% aqueous potassium carbonate (25 mL), the organic layerseparated, dried over sodium sulfate, filtered and concentrated underreduced pressure. Column chromatography of the resulting solid (silicagel, EtOAc/hexanes) gave a 52% yield of compound 169 as a white solid.m.p. 117-118° C.; ¹H NMR (DMSO-d₆) S (ppm) 7.72-7.60 (m, 2H), 7.42 (dd,1H, J=7.6 Hz, 1.3 Hz), 7.31 (d, 1H, J=7.0 Hz), 6.95 (t, 1H, J=9.3 Hz),6.82 (m, 2H), 4.27 (m, 1H), 1.33 (s, 9H), 1.28 (d, 3H, J=7.2 Hz);m/z=436 (M+H). The reaction described above and compound 169 are shownbelow.

2-(1-amino-ethyl)-5-chloro-3-(3, 5-difluoro-phenyl)-3H-quinazolin-4-one(170)

Step D: A solution of compound 169 (1.68 mmol) in piperidine (2 mL) wasstirred for 3 h at ambient temperature. Evaporation of the reactionmixture to dryness under high vacuum gave a yellow foam. This foam wasdissolved in a 4M solution of hydrogen chloride in 1,4-dioxane (4 mL)and stirred for 17 h at ambient temperature. The reaction mixture wasthen concentrated to dryness, basified with 10% aqueous potassiumcarbonate (40 mL) and extracted with MTBE (3×20 mL). Combining theorganic extracts, drying over sodium sulfate and concentrating todryness afforded a solid residue. This residue was dissolved ind-chloroform (5 mL), and warmed for 15 h at 50° C. After cooling toambient temperature, the reaction mixture was washed with water (3×10mL), dried over sodium sulfate and evaporated to dryness, affording a98% yield of compound 170 as a white solid. m.p. 200-202° C.; ¹H NMR(CDCl₃) δ (ppm) 7.65 (m, 2H), 7.49 (m, 1H), 7.01 (t, 1H, J=6.6 Hz), 6.89(m, 2H), 3.68 (m, 1H), 1.33 (d, 3H, J=6.6 Hz), 1.25 (s, 2H); m/z=336(M+H). The reaction described above and compound 170 are shown below.

5-chloro-3-(3,5-difluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(166)

Step E: Compound 166 was prepared by reacting compound 170 in accordingto the procedure for the preparation of compound 107 (final procedure).ESI-MS m/z 454.3 (MH⁺). The reaction described above and compound 166are shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-5-chloro-3-(3,5-difluoro-phenyl)-3H-quinazolin-4-one(171)

Compound 171 was prepared following the general procedure for compound161 (steps A-E), but 2-tert-butoxycarbonylamino-butyric acid2,5-dioxo-pyrrolidin-1-yl ester was substituted forN-tert-butyloxycarbonyl-D,L-alanine N-hydroxysuccinimide ester in stepB, and 2-amino-6-bromopurine was substituted for 6-bromopurine in stepE. ESI-MS m/z 454.3 (MH⁺). The structure of compound 171 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-ethyl]-5-chloro-3-(3,5-difluoro-phenyl)-3H-quinazolin-4-one(172)

Compound 172 was prepared following the general procedure for compound161 (steps A-E), but 2-amino-6-bromopurine was substituted for6-bromopurine in step E. The structure of compound 172 is shown below.

3-(3,5-difluoro-phenyl)-6-fluoro-2-[1-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one(173)

Compound 173 was prepared following the general procedure for compound161 (steps A-E), but 2-amino-5-fluorobenzoic acid was substituted for2-amino-6-chlorobenzoic acid in step A. ESI-MS m/z 438.2 (MH⁺). Thestructure of compound 173 is shown below.

5-chloro-3-(2,6-difluoro-phenyl)-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one(174)

Compound 174 was prepared following the general procedure for compound161 (steps A-E), but 2,6-difluoroaniline was substituted for aniline instep A, and 2-tert-butoxycarbonylamino-butyric acid2,5-dioxo-pyrrolidin-1-yl ester was substituted forN-tert-butyloxycarbonyl-D,L-alanine N-hydroxysuccinimide ester in stepB. ESI-MS m/z 468.2 (MH⁺). The structure of compound 174 is shown below.

2-[1-(2-amino-9H-purin-6-ylamino)-propyl]-5-chloro-3-(2,6-difluoro-phenyl)-3H-quinazolin-4-one (175)

Compound 175 was prepared following the general procedure for compound161 (steps A-E), but 2,6-difluoroaniline was substituted for aniline instep A, 2-tert-butoxycarbonylamino-butyric acid2,5-dioxo-pyrrolidin-1-yl ester was substituted forN-tert-butyloxycarbonyl-D,L-alanine N-hydroxysuccinimide ester in stepB, and 2-amino-6-bromopurine was substituted for 6-bromopurine in stepE. ESI-MS m/z 483.2 (MH⁺). The structure of compound 175 is shown below.

Example 13 Compound Preparation

Compounds having general formula I (shown above) have been prepared inaccordance with steps A-G of the synthetic scheme entitled “Procedure L”shown below.

5-methyl-3-phenyl-2-[1-(9H-purin-6-yloxy)-ethyl]-3H-quinazolin-4-one(176)

Compound 176 was prepared according to the procedures set forth in stepsA-G below.

acetic acid 1-(3-methyl-2-phenylcarbamoyl-phenylcarbamoyl)-ethyl ester(177)

Step A: (S)-2-Acetoxypropionyl chloride (5.469 g, 36.32 mmol) was addedto a solution of compound 15 (6.788 g, 30 mmol) in dichloromethane (150mL). A precipitate immediately formed. The reaction was stirred for 25 hand the precipitate was filtered off. The filtrate was washed withsaturated sodium bicarbonate solution (3×50 mL) and dried (MgSO₄).Filtration and concentration of the filtrate gave a brown solid (7.6 g).Purification by flash chromatography (1:2 EtOAc:hexanes->EtOAc->10:1EtOAc:MeOH) followed by recrystallization of the impure fractions fromEtOAc:hexanes gave compound 177 as a solid. ESI-MS m/z=341 (MH⁺). Thereaction described above and compound 177 are shown below.

acetic acid 1-(5-methyl-4-phenylimino-4H-benzo[d][1,3]oxazin-2-yl)-ethylester (178)

Step B: Compound 177 (0.34 g, 1.0 mmol) was dissolved in dichloromethane(25 mL). Triphenylphosphine (1.311 g, 5 mmol) was added to the solution,followed by iodine (1.269 g, 5 mmol) and DIEA (1.9 mL, 11 mmol). Thereaction was capped and stirred for 4 days. The reaction was quenched byaddition of saturated aqueous sodium bicarbonate solution (25 mL). Theorganic layer was separated, dried (MgSO₄), filtered, and the filtrateconcentrated under reduced pressure to give a dark brown gum (2.886 g).Purification by flash chromatography (CH₂Cl₂) gave the imino-1,3-oxazinecompound 178 as a yellow oil. ESI-MS m/z=323 (MH⁺). The reactiondescribed above and compound 178 are shown below.

acetic acid1-methyl-2-(3-methyl-2-phenylcarbamoyl-phenylimino)-2-piperidin-1-yl-ethylester (179)

Step C: Piperidine (1 mL) was added to compound 178 (0.161 g, 0.5 mmol)and the reaction mixture was stirred for 19.5 h. The reaction mixturewas then concentrated under reduced pressure to give a yellow gum.Trituration with 1:4 EtOAc:hexanes gave a small amount of compound 179(0.041 g). Flash chromatography (1:4 EtOAc:hexanes) of the filtrate gaveonly a partially separable mixture of the expected products, theacetoxyquinazolinone compound 179 and the hydroxyquinazolinone (totalmass 0.122 g). ESI-MS m/z=408 (MH⁺). The reaction described above andcompound 179 are shown below.

acetic acid1-(5-methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-ethyl ester(180)

Step D: Compound 179 (0.037 g, 0.09 mmol) was dissolved in acetonitrile(10 mL) and the reaction mixture was heated at reflux for 3 h. Thesolvent was removed under reduced pressure and the residue dissolved ina mixture of ethyl acetate (10 mL) and 1M HCl (5 mL). After separatingthe aqueous layer, the organic layer was washed with additional 1M HCl(2×5 mL), saturated sodium bicarbonate solution (3×5 mL), water (2×5 mL)and saturated brine (5 mL). The solution was dried (MgSO₄), filtered,and the filtrate concentrated under reduced pressure to give compound180. ESI-MS m/z=323 (MH⁺). The product had nearly totally racemized atthis point (chiral purity was 46:54 S:R). The reaction described aboveand compound 180 are shown below.

2-(1-hydroxy-ethyl)-5-methyl-3-phenyl-3H-quinazolin-4-one (181)

Step E: Compound 180 (0.011 g, 0.034 mmol) was dissolved in methanol (2mL), and potassium carbonate (0.012 g, 0.085 mmol) was added. Thereaction mixture was stirred for 20 min, and then water (20 mL) wasadded. The mixture was extracted with ethyl acetate (3×10 mL) and thecombined organic extracts were washed with saturated brine (10 mL). Theorganic solution was dried (MgSO₄), filtered and concentrated underreduced pressure to give compound 181. ESI-MS m/z=281 (MH⁺). Thereaction described above and compound 181 are shown below.

5-methyl-3-phenyl-2-(1-[9-(2-trimethylsilanyl-ethoxymethyl)-9H-purin-6-yloxy]-ethyl)-3H-quinazolin-4-one(182)

Step F: A solution of compound 181 (0.069 g, 0.25 mmol) in THF (5 mL)was treated with sodium hydride (0.007 g, 0.27 mmol) and stirred for 10min. A solution of intermediate compound 13 (0.077 g, 0.27 mmol) in THF(1 mL) was added to the reaction mixture. The flask originallycontaining the intermediate compound 13 was washed with additional THF(1 mL) and the washings were also added to the reaction mixture. Thereaction was allowed to proceed, and additional sodium hydride (0.005 g,0.21 mmol) was added at 21.5 h and 23 h. The reaction was quenched aftera total of 24 h by addition of saturated ammonium chloride solution (5mL). The mixture was extracted with dichloromethane (3×5 mL), and thecombined organic extracts were dried over MgSO₄. After filtration, thefiltrate was concentrated under reduced pressure and the residue waspurified by flash chromatography (1:1 EtOAc:hexanes->3:2 EtOAc:hexanes)to give compound 182. ESI-MS m/z=529 (MH⁺). The reaction described aboveand compound 182 are shown below.

5-Methyl-3-phenyl-2-[1-(9H-purin-6-yloxy)-ethyl]-3H-quinazolin-4-one(176)

Compound 182 (0.053 g, 0.1 mmol) was dissolved in a mixture of methanol(2 mL) and 4M HCl (2 mL). The mixture was stirred and heated to 40° C.for 3 h. The reaction mixture was removed from the heat source andallowed to cool. The reaction mixture was then filtered through a plugof GFA (glass fiber) filter paper and the filtrate concentrated to onlyremove the methanol. The residue was adjusted to pH 10 by addition of10% potassium carbonate solution. The resulting solid was collected byfiltration and purified by RP-HPLC (C18 Luna column, 10×250 mm, 4.7mL/min, 10-90% CHsCN in water in 18 min, with all solvents containing0.05% formic acid) to give compound 176 as a fluffy white solid afterlyophilization. ¹H NMR (400 MHz, d₆-DMSO) δ 13.40, br s, 1H; 8.40, s,1H; 8.35, s, 1H; 7.66, t, J=7.8 Hz, 1H; 7.48-7.58, m, 4H; 7.31-7.36, m,2H; 7.20-7.23, m, 1H; 5.65, q, J=6.6 Hz, 1H; 2.73, s, 3H; 1.65, d, J 6.6Hz, 3H. ESI-MS m/z=399 (MH⁺). The reaction described above and compound176 are shown below.

Example 14 Biochemical Assays of PI3K Potency and Selectivity

Biochemical Assay using 20 μM ATP

Using the method described in Example 2, compounds of the invention weretested for inhibitory activity and potency against PI3Kδ, and forselectivity for PI3Kδ versus other Class I PI3K isogymes. In Table IC₅₀values (μM) are given for PI3Kδ (“Delta”), and may be calculated for theother isoforms using the ratios of IC₅₀ values discussed below. Toillustrate selectivity of the compounds, the ratios of the IC₅₀ valuesof the compounds for PI3Kα, PI3Kβ, and PI3Kγ relative to PI3Kδ aregiven, respectively, as “Alpha/Delta Ratio,” “Beta/Delta Ratio,” and“Gamma/Delta Ratio.”

The initial selectivity assays were performed identically to theselectivity assay protocol in Example 2, except using 100 μL Ecoscintfor radiolabel detection. Subsequent selectivity assays were donesimilarly using the same 3× substrate stocks except they contained 0.05mCi/mL γ[³ZP]ATP and 3 mM PIP2. Subsequent selectivity assays also usedthe same 3× enzyme stocks, except they now contained 3 nM of any givenPI3K isoform.

For all selectivity assays, the test compounds were weighed out anddissolved into 10-50 mM stocks in 100% DMSO (depending on theirrespective solubilities) and stored at −20° C. Compounds were thawed (toroom temperature or 37° C.), diluted to 300 μM in water from which a3-fold dilution series into water was done. From these dilutions, 20 μLwas added into the assay wells alongside water blanks used for theenzyme (positive) control and the no enzyme (background) control. Theremainder of the assay was performed essentially according to theselectivity assay protocol in Example 2.

TABLE 1 Human PMN Delta Alpha- Beta- Gamma- Elastase Human B IC₅₀ DeltaDelta Delta EC₅₀ Lymphocyte (nM) Ratio Ratio Ratio (nM) EC₅₀ (nM)Compound 12 662 78 67 382 1.6 117 Compound 16 608 74 66 84 4.6 98Compound 9 248 49 21 119 6.1 107 Compound 45 250 72 38 298 30.7 174Compound 26 721 94 62 584 93

Example 15 Cell-Based Assay Data for Inhibitors of PI3K8 Activity

Using the methods described in Example 2, compounds of the inventionwere tested for inhibitory activity and potency in an assay ofneutrophil (PMN) elastase release. Data from these assays are set forthin Table 1. In Table 1, the values shown are effective concentrations ofthe compound (EC₅₀; μM)

All publications and patent documents cited in this specification areincorporated herein by reference for all that they disclose.

While the present invention has been described with specific referenceto certain preferred embodiments, further modifications can be practicedwithin the scope of the invention as it is defined in the claims below.Accordingly, no limitations should be placed on the invention other thanthose specifically recited in the claims.

1-26. (canceled) 27: A compound of formula (110)

28: A composition comprising the compound of formula (110) according toclaim
 27. 29: A method of preparing a compound of formula (110)according to claim 27, comprising:

reacting 2-fluoro-6-nitrobenzoic acid with aniline to provide a compoundof formula (108)

reacting the compound of formula (108) with Boc-protected 2-aminobutyricacid to provide a compound of formula (109)

and cyclizing the compound of formula (109) to provide the compound offormula (110) according to claim
 27. 30: The method of claim 29, wherein2-fluoro-6-nitrobenzoic acid is reacted with aniline in the presence ofoxalyl chloride. 31: The method of claim 29, wherein the compound offormula (108) is reacted with Boc-protected 2-aminobutyric acid in thepresence of thionyl chloride. 32: The method of claim 31, wherein thecompound of formula (108) is further reacted with Boc-protected2-aminobutyric acid in the presence of triethylamine. 33: The method ofclaim 29, wherein the compound of formula (109) is cyclized in thepresence of zinc. 34: The method of claim 29, further comprisingdeprotecting the compound of formula (110)

35: The method of claim 34, wherein the compound of formula (110) isdeprotected with an acid.